Gas-filled microvesicles composition for contrast imaging

ABSTRACT

The present invention relates to a new composition comprising gas-filled microvesicles for contrast imaging which are particularly suitable for providing an effective echo response to at least two selected ultrasound waves having different frequencies. Said composition preferably comprises at least two different preparations of gas-filled microvesicles having respective peaks of non-linear echographic response differing by at least 2 MHz to each other, and preferably have respective size distributions with different median sizes. In particular, said preparations preferably have size distributions with respective DV50 values differing from each other by at least 0.5 μm, more preferably at least 1.0 μm. Alternatively, said composition has a volume size distribution showing a value of Bowley skewness of 0.16 or higher. According to a preferred embodiment, at least 95% of the total volume of gas contained in said microvesicles, calculated on the population of microvesicles up to a diameter of 10 μm, is contained in microvesicles having a diameter of 8 micron or less.

FIELD OF THE INVENTION

The present invention relates to a gas-filled microvesicles compositionsuitable for use as contrast agents in diagnostic and/or therapeuticimaging, to diagnostic/therapeutic imaging methods comprising the use ofsaid composition and to a preparation method of said composition.

BACKGROUND OF THE INVENTION

Rapid development of ultrasound contrast agents in recent years hasgenerated a number of different formulations, which are useful inultrasound contrast imaging of organs and tissue of a human or animalbody. These agents are designed to be used primarily as intravenous orintra-arterial injectables in conjunction with the use of medicalechographic equipment which employs for example, B-mode image formation(based on the spatial distribution of backscatter tissue properties) orDoppler signal processing (based on Continuous Wave or pulsed Dopplerprocessing of ultrasonic echoes to determine blood or liquid flowparameters).

A class of injectable formulations useful as ultrasound contrast agentsincludes suspensions of gas bubbles having a diameter of a few micronsdispersed in an aqueous medium.

Of particular interest are gas bubbles which are stabilized by means ofsuitable additives such as, for example emulsifiers, oils, thickeners orsugars, or by entrapping or encapsulating the gas or a precursor thereofin a variety of systems. These stabilized gas bubbles are generallyreferred to in the art as “microvesicles”, and may be divided into twomain categories.

A first category of stabilized bubbles or microvesicles is generallyreferred to in the art as “microbubbles” and includes aqueoussuspensions in which the bubbles of gas are bounded at the gas/liquidinterface by a very thin envelope (film) involving a stabilizingamphiphilic material disposed at the gas to liquid interface.Microbubble suspensions are typically prepared by contacting powderedamphiphilic materials, e.g. freeze-dried preformed liposomes orfreeze-dried or spray-dried phospholipid solutions, with air or othergas and then with an aqueous carrier, while agitating to generate amicrobubble suspension which can then be administered, preferablyshortly after its preparation.

Examples of aqueous suspension of gas microbubbles and preparationthereof are disclosed, for instance, in U.S. Pat. Nos. 5,271,928,5,445,813, 5,413,774, 5,556,610, 5,597,549, 5,827,504, WO 97/29783 andin co-pending International Patent Application PCT/IB04/00243, which arehere incorporated by reference in their entirety.

A second category of microvesicles is generally referred to in the artas “microballoons” or “microcapsules” and Includes suspensions in whichthe bubbles of gas are surrounded by a solid material envelope of alipid or of natural or synthetic polymers. Examples of microcapsules andof the preparation thereof are disclosed, for instance, in U.S. Pat. No.5,711,933 and U.S. Pat. No. 6,333,021, herein incorporated by referencein their entirety.

Microvesicles preparations are characterized, among other factors, alsoby their respective mean size and size distribution (which gives anIndication on how the microvesicle population is scattered around themean size). Size-distributions of microvesicles preparations can ingeneral be assimilated to a Gaussian-like distribution, centred on themean size value thereof.

Contrast imaging is based on the ability of gas-filled microvesicles toresonate when hit by an ultrasound wave emitted by an ultrasound probeat a certain frequency, thus reflecting a corresponding echo signalwhich is detected by the ultrasound probe and then imaged. As the echoresponse of a contrast agent is rather peculiar with respect to the echoresponse of tissues or organs itself, the contrast agent contained inthe vessels can be easily imaged with respect to the surrounding tissueor organ. The resonance capacity of a gas-filled microvesicle depends,among other factors, also from the compatibility of its size with thefrequency of the transmitted radiation. As a general indication, smallermicrovesicles resonate at higher frequencies, while larger microvesiclesresonate at lower frequencies. In addition, the intensity of a reflectedecho is in general proportional to the concentration of microvesicleshaving said predetermined compatible dimensions, said concentrationbeing for instance expressed as the total volume of gas entrapped insaid microvesicles.

The Applicant has now observed that, for a specific contrast agent, itis possible to define a preferred size range and a corresponding sizedistribution which is suitably responsive to a determined transmissionfrequency. As observed by the Applicant, at low frequencies (e.g. fromabout 1.5 to about 3.5 MHz), said size distribution typically has arelatively large median diameter (e,g. D_(V50) of about 4 μm) and is ingeneral relatively broad; this observation is consistent with the factthat conventional broadly distributed gas-filled microvesicles can ingeneral be employed for the contrast imaging at these low frequencies,as a sufficiently large number of microvesicles are available forresonating when hit by the selected low frequency ultrasound wave. Onthe other side, at higher frequencies (e.g. 5 MHz or higher), the sizedistribution of suitably responsive microvesicles substantially narrows.In addition, said narrow distribution is generally associated with acorresponding relatively smaller D_(V50) value (e.g. from about 1 to 2.5μm), in accordance with the fact that small microvesicles resonate athigher frequency. This observation is also consistent with the fact thatconventional broadly distributed microvesicle preparations are ingeneral much less responsive at high frequency contrast imaging, as thefraction of small dimensions microvesicle contained therein isrelatively low. Thus, when using high transmission frequencies forultrasound imaging, suitably calibrated gas-filled microvesiclepreparations having relatively narrow size distributions with relativelysmall median dimensions (D_(V50), in particular) shall be employed foran effective contrast imaging, said preparations being however not aseffective when used at low transmission frequencies.

In general, relatively low transmission frequencies (e.g. 0.5-2 MHz) areemployed for echographic analysis in deep body regions, such as forcardiac applications, while relatively high transmission frequencies(e.g. 5-7 and up to 10-15 MHz) are generally employed for abdominal(e.g. kidney, liver etc.) or superficial analysis (e.g. ophthalmology,breast analysis etc.). Higher transmission frequencies (e.g. 15-20 MHzand up to 80 MHz) can also be employed for specific applications, forinstance in intravascular ultrasound imaging.

The Applicant has now found a new composition suitable for providing aneffective echo response to at least two selected ultrasound waves havingdifferent frequencies. As observed by the Applicant, said effective echoresponse can be obtained by suitably tailoring the size distribution ofa gas-filled microvesicles preparation. Advantageously, said preparationcomprises an effective amount of microvesicles having a relatively smallsize, being thus responsive to a respective relatively high selectedtransmission frequency, and an effective amount of microvesicles with arelatively larger size, responsive to a respective relatively lowerselected transmission frequency, said effective amount of large sizemicrovesicles being nevertheless sufficiently low so as to notexcessively attenuate the response of the small size microvesicles atthe selected high transmission frequency.

International Patent application WO 98/32468 discloses compositionscomprising two or more types of gas containing microparticles havingdifferent susceptibility to ultrasonic pressure. Preferred compositionare those comprising a first type of microparticles with a relativelysoft encapsulating shell (such as microbubbles with phospholipid shells)and a second type of microparticles with relatively hard encapsulatingmaterial (such as polymer or protein shelled microcapsules). Inparticular, example 1 of said patent discloses mixtures of microbubblescomprising hydrogenated egg phosphatidylserine with microcapsulescontaining a polymer comprising repeating units of formula:—O—(CH₂)_(a)—CO—O—CH(CH₃)—O—CO—(CH₂)_(a)—CO—O(CH₂)_(b)—CO—

where a is an integer from 9 to 19 and b is an integer from 1 to 8.

WO01/68150 discloses microcapsules having a stabilizing envelopecomprising a polyalkylcyanoacrylate polymer.

SUMMARY OF THE INVENTION

An aspect of the invention relates to a composition for diagnosticand/or therapeutic imaging which comprises at least two differentpreparations of gas-filled microvesicles, wherein said at least twodifferent preparations have respective peaks of non-linear echographicresponse differing by at least 2 MHz to each other, preferably by atleast 3 MHz.

According to a preferred embodiment, said respective peaks of non-linearechographic response are from about 1.5 to about 10 MHz. Preferably,said composition comprises a first preparation of microvesicles with apeak of non-linear echographic response of 3 MHz or lower, morepreferably from 1.5 to 3 MHz, and a second preparation of microvesicleswith a peak of non-linear echographic response of 5 MHz or higher, morepreferably from 5 to 10 MHz.

According to a further preferred embodiment, said different preparationsof microvesicles have respective size distributions with differentmedian diameter. Preferably, said size distributions are defined by arespective at least first and at least second median diameter in volume(D_(V50)), said first and second D_(V50) differing from each other by avalue of at least 0.5 μm, more preferably at least 1.0 μm and even morepreferably of at least 1.5 μm. Preferably, at least 95% of the totalvolume of gas contained in said microvesicles, calculated on thepopulation of microvesicles up to a diameter of 10 μm, is contained inmicrovesicles having a diameter of 8 micron or less. According to afurther preferred embodiment, the microvesicles of at least one of saidat least two sets have a size distribution defined by a respective ratiobetween said mean diameter in volume and a corresponding mean diameterin number (D_(V)/D_(N)), at least one of said sets of gas-filledmicrovesicles having a D_(V)/D_(N) ratio of from 1.2 to 3, preferably offrom 1.2 to 2.

Another aspect of the invention relates to a composition comprisinggas-filled microvesicles for use in diagnostic imaging, wherein the sizedistribution of said gas-filled microvesicles has a Bowley skewness of0.16 or higher. Preferably, at least 95% of the total volume of gascontained in said microvesicles is contained in microvesicles having adiameter of 8 micron or less. In the present description and claims theBowley skewness is calculated on the experimental plot of the volumesize distribution of a gas-filled microvesicle preparation orcomposition, in the population of microvesicles having a diameter up to8 μm.

Another aspect of the invention relates to a method for conferring, to acomposition comprising gas-filled microvesicles having a peak ofnon-linear echographic response to a first transmission frequency, anenhanced echographic response to a second transmission frequency, whichcomprises admixing said composition with a second composition ofgas-filled microvesicles having a respective peak of non-linearechographic response to said second frequency. Preferably, said firstand second frequency differ by at least 2 MHz to each other, morepreferably by at least 3 MHz.

A further aspect of the invention relates to a method of manufacturingan ultrasound contrast agent having an effective echographic response toat least two different transmission frequencies, which comprisesadmixing at least two different preparations of gas filled microvesicleshaving respective peaks of non-linear echographic response differing byat least 2 MHz to each other, preferably by at least 3 MHz. Preferably,said at least two different preparations of gas-filled microvesicleshave respective different size distributions adapted for an effectiveresponse to said at least two different transmission frequencies.

A further aspect of the invention relates to a method of diagnosticand/or therapeutic imaging which comprises administering to a patient aneffective amount of a composition as above defined.

DRAWINGS

FIG. 1 shows a schematic representation of comparative microvesiclessize distributions.

FIG. 2 shows a schematic representation of a size distribution of acomposition of the invention compared to the schematic broad sizedistribution of a conventional preparation.

FIG. 3 shows a schematic method for calculating an optimal sizedistribution for a selected transmission frequency.

FIG. 4 shows a calculated optimal size distribution.

FIG. 5a shows the size distribution of experimental preparationsaccording to example 1, including combined preparation CM1A. FIG. 5bshows the size distribution of experimental preparations according toexample 1, including combined preparation CM1B. FIG. 5c shows the sizedistribution of experimental preparations according to example 1,including combined preparation CM1C.

FIG. 6a shows the size distribution of experimental preparationsaccording to example 2, including combined preparation CM2A. FIG. 6bshows the size distribution of experimental preparations according toexample 2, including combined preparation CM2B. FIG. 6c shows the sizedistribution of experimental preparations according to example 2,including combined preparation CM2C.

FIG. 7 shows the size distribution of experimental preparationsaccording to example 3, including combined preparations CM3A and CM3B.

FIG. 8 shows the size distribution of experimental preparationsaccording to example 4, including combined preparation CM4B.

FIG. 9 shows the size distribution of experimental combined preparationaccording to example 5 and preparations M4a and M4b of example 4.

FIG. 10 shows an illustrative representation of the parameters definingthe Bowley Skewness.

FIG. 11 shows a schematic representation of the experimental setup forthe measurement of the echo-power response of microbubble preparations.

FIG. 12a shows the echo-power response of an experimental microbubblepreparation at a transmission frequency of 2 MHz. FIG. 12b shows theecho-power response of an experimental microbubble preparation at atransmission frequency of 10 MHz.

FIG. 13 shows the size distribution of a composition of the inventioncompared to a commercial ultrasound contrast agent.

DETAILED DESCRIPTION

The dimensions and size distribution of gas-filled microvesicles can becharacterized by a number of parameters, the most frequently used beingthe mean diameter in number D_(N) and the mean diameter in volume D_(V).While diameter in number provides an indication of the mean numberdimension of the microvesicles, the diameter in volume providesinformation on how the total volume of gas entrapped in themicrovesicles is distributed among the population thereof. Additionaluseful parameters for characterizing a population of gas-filledmicrovesicles are the D_(V50), D_(V90) or D_(V95) diameters. Theseparameters indicate the percentage of gas (50, 90 or 95%, respectively)which is entrapped in microvesicles having a diameter equal to or lowerthan said value. Thus, for instance, D_(V90)=10 μm means that 90% of thetotal volume of gas of the microvesicle preparation referred to iscontained in microvesicles having a diameter of 10 μm or less TheD_(V50) value defines the median diameter in volume of a sizedistribution. While theoretically mono-sized microvesicles would showidentical D_(N) and D_(V) or D_(V50) values, a narrow or broad sizedistribution in experimental preparations will determine a correspondingsmall or large difference, respectively, between the D_(N) and D_(V)values and, accordingly, with a corresponding variation of theD_(V)/D_(N) ratio. The value of the D_(V)/D_(N) ratio can thus be usedto estimate how much the size distribution of a certain population ofgas-filled microvesicles is dispersed around its mean value; in general,the broader the size distribution, the larger the value of theD_(V)/D_(N) ratio. Thus, for example, populations containing primarilysmall microvesicles (e.g. microvesicles with a diameter around 2 μm)with substantially no large bubbles (for instance bubbles with adiameter above 8 μm) will show a D_(V) value close to the D_(N) value,with a correspondingly relatively low D_(V)/D_(N) ratio. Conversely,populations containing primarily small microvesicles with nevertheless asmall percentage of large bubbles will show a higher D_(V) value, with acorrespondingly higher D_(V)/D_(N) ratio. In general, a population ofmicrovesicles showing a D_(V)/D_(N) ratio of less than about 2 can beconsidered as being narrowly distributed; these microvesicles can alsobe referred to as “calibrated” microvesicles. On the other side, apopulation of microvesicles showing a D_(V)/D_(N) ratio of about 3 ormore can in general be considered as having a broad distribution.

FIGS. 1 and 2 illustrate an example of the advantages of an aspect ofthe present invention, whereby at least two different gas-filledmicrovesicles preparations are combined to obtain an effectiveechographic response to at leas two different transmission frequencies.In FIG. 1, solid line 11 shows a schematic representation of thenormalized distribution of the gas volume with respect to microvesicles'size in a typical population of microvesicles with broadsize-distribution (BM). The dashed line 12 shows a schematicrepresentation of the normalized distribution of the gas volume in afirst population of narrowly distributed microvesicles (NM1), having aD_(V50) value of 1.9 μm, said size distribution being adapted for aneffective response to a first transmission frequency f₁. The dotted line13 shows a schematic representation of the normalized distribution ofgas volume in a second population of relatively less narrowlydistributed microvesicles (NM2), having a D_(V50) value of 4.1 μm, saidsize distribution being adapted for an effective response to a secondtransmission frequency f₂. For the sake of clarity, a symmetricalGaussian distribution has been adopted for the schematic representationsof the size distributions of BM, NM1 and NM2 whilst, as explained in thefollowing of the specification, experimental size distributions patternsmay in general be more or less distorted with respect to said symmetricdistribution.

When a selected transmission frequency f1 hits the microvesicles of BMor NM1, a respective portion of said microvesicles having dimensionscompatible with said frequency (i.e. mainly those included in the sliceS1 defined around the size compatible with the transmission frequency)will resonate and reflect an echo signal with a determined intensity.The intensity of the reflected echo signal will be substantiallyproportional to the volume of gas contained in the respective areadefined by S1 under 12 (NM1) or 11 (BM). As inferable from FIG. 1, thevolume of gas comprised in the area of S1 defined under 12 is muchlarger than the corresponding volume of gas comprised in the same slicedefined under 11, thus resulting in a more intense reflected echo andfinally a better image enhancement. Similar observations can be madewhen a second lower transmission frequency f2 hits the compatiblemicrovesicles in the slice S2 of BM or NM2.

In FIG. 2, the solid line 21 shows a schematic representation of thenormalized distribution of the gas volume in a combined composition (CC)obtained by mixing NM1 and NM2 in a 1:1 volume ratio. Dotted line 22shows the normalized size distribution of the gas volume inmicrovesicles of the previous preparation BM. As inferable from thisfigure, a combined composition according to an aspect of the inventionallows an effective volume of gas to be available in microvesicles ofsizes compatible with the two transmission frequencies f1 and f2. On thecontrary, the use of a conventional BM preparation will be restricted incombination with the sole transmission frequency f2. In the particularcase, the higher amount of microvesicles compatible with frequency f1 inthe CC with respect to BM will allow a corresponding higher echoresponse to be generated. This higher echo response will result in aneffective echo contrast imaging also in the presence of a relativelylarge amount of larger microvesicles which, further of not beingresponsive to the selected frequency, primarily contribute to theattenuation of the signal, both of the transmitted and of the reflectedone. It is worth to note that, whilst the response of BM in therespective area of slice S1 could in theory be increased by increasingthe total volume of gas thereof (i.e. using higher amounts of the BMpreparation), this increase may however not be desirable in thepractice. A first reason is that it is in general preferred to keep theconcentration of a contrast agent as low as possible (consistently withan acceptable imaging enhancement), in order to avoid any possible sideeffects thereof. The other reason is that an increase of the totalvolume of gas in BM will determine a corresponding increase of thefraction of large microvesicles, which will determine an unacceptableattenuation of the ultrasound signal.

As observed by the Applicant, the size distribution pattern of acomposition of the invention (when plotted on a graph having themicrovesicles size as abscissa and the normalized volume percentage asordinate) is rather peculiar with respect to a typical size distributionpattern defined by a single microvesicle preparation. In general, thislatter can in fact be assimilated to a substantially Gaussiandistribution, also referred to as Gaussian-like distribution, with agenerally slightly dispersed distribution in its right half portion.Deviations of distributions from the symmetrical Gaussian distribution,i.e. dispersion, can be represented by means of conventional parameters,such as the “skewness”. As know in the art, the skewness is a measure ofsymmetry, or more precisely, the lack of symmetry of a distribution ordata set. Robust measures for skewness can be found in literature. Auseful coefficient of skewness is the “Bowley coefficient of skewness”(Elements of statistics, New York: Charles Scribner's Sons, 1920), alsoknown as “quartile skewness coefficient”, which is defined by thefollowing:

$\begin{matrix}{{{BS} = \frac{Q_{3} - {2Q_{2}} + Q_{1}}{Q_{3} - Q_{1}}},} & (1)\end{matrix}$where Q_(i) is the i^(th) quartile of the distribution. Thus, asillustrated in FIG. 10, in the case of the gas volume distribution of amicrovesicle preparation, Q₁ corresponds to the larger diameter of themicrovesicles entrapping up to 25% of the total volume of entrapped gas,Q₂ corresponds to the larger diameter of the microvesicles entrapping upto 50% of the total volume of entrapped gas and Q₃ corresponds to thelarger diameter of the microvesicles entrapping up to 75% of the totalvolume of entrapped gas. It can be seen from equation (1) and FIG. 10that for any symmetric distribution BS=0. The denominator, Q₃−Q₁,re-scales the coefficient so that the maximum value for BS (i.e. 1)represents extreme right skewness, while the minimum value for BS (i.e.−1) represents extreme left skewness.

The Applicant has now observed that compositions suitable for being usedto at least two different transmission frequencies have in general arather pronounced dispersion in their respective right half portion.Thus, according to another aspect of the invention, a compositionaccording to the invention have BS values higher than 0.16, preferablyof at least 0.18 or higher and more preferably of at least 0.20 orhigher, up to e.g 0.40. For the purposes of characterizing thecompositions according to the invention, the values of the Bowleyskewness are herein calculated in the range of sizes from 0 μm to 8 μm,in order to avoid any possible miscalculation determined by anundesirable contribution of few uncontrolled large sized microvesicles.All the values of BS given in the present specification and claims arethus referred to a calculation including only microvesicles up to adiameter of 8 μm. Preferably, the stabilizing envelope of themicrovesicles of the composition having said BS values does not comprisea polyalkylcyanoacrylate polymer.

Rather peculiarly, in some cases the central portion of the distributionpattern of a combined composition is substantially flat, while in otherparticular cases a local minimum can be observed in said central portion(such as in the schematic distribution illustrated in FIG. 2).

According to a preferred embodiment of the invention, compositions withthe above values of BS can advantageously be obtained by combining twoor more different gas-filled microvesicles preparations. In a preferredembodiment of the invention, in order to minimize the undesirableattenuation effects of large size microvesicles, in particular whenoperating at rather high transmission frequencies, at least 95% of thetotal volume of gas (D_(V95)) contained in a gas-filled microvesiclecomposition of the Invention is contained in microvesicles having adiameter of 8 micron or less. For the purposes of the invention, inorder to determine said D_(V95) value, only microvesicles with adiameter up to 10 μm are taken into consideration for the calculation.In particular, the D_(V95) value of the combined composition is 7 μm orlower, preferably 6.5 μm or lower and more preferably 6 μm or lower,down to e.g. about 4 μm.

Gas-filled microvesicles suitable for preparing a combined compositionaccording to the invention can be any kind of microvesicles known in theart, such as gas-filled microbubbles or gas-filled microcapsules,typically contained as a suspension in a physiologically acceptableliquid carrier. Preferably, said microvesicles are microbubbles.

The term “physiologically acceptable” includes within its meaning anycompound, material or formulation which can be administered, in aselected amount, to a patient without negatively affecting orsubstantially modifying its organism's healthy or normal functioning(e.g. without determining any status of unacceptable toxicity, causingany extreme or uncontrollable allergenic response or determining anyabnormal pathological condition or disease status).

Microbubbles

Gas-filled microbubbles as defined herein comprise bubbles of gasdispersed in an aqueous suspension which are stabilized by a thinenvelope comprising an amphiphilic compound disposed at the gas toliquid interface. Said stabilizing envelope, sometimes referred to as an“evanescent envelope” in the art, has in general a thickness of lessthan 5 nm, typically of about 2-3 nm, thus often amounting to asubstantially monomolecular layer.

The amphiphilic compound included in the microbubbles' envelope can be asynthetic or naturally-occurring biocompatible compound and may include,for example a film forming lipid, in particular a phospholipid. Examplesof amphiphilic compounds include, for instance, phospholipids;lysophospholipids; fatty acids, such as palmitic acid, stearic acid,arachidonic acid or oleic acid; lipids bearing polymers, such as chitin,hyaluronic acid, polyvinylpyrrolidone or polyethylene glycol (PEG), alsoreferred as “pegylated lipids”; lipids bearing sulfonated mono- di-,oligo- or polysaccharides; cholesterol, cholesterol sulfate orcholesterol hemisuccinate; tocopherol hemisuccinate; lipids with etheror ester-linked fatty acids; polymerized lipids; diacetyl phosphate;dicetyl phosphate; ceramides; polyoxyethylene fatty acid esters (such aspolyoxyethylene fatty acid stearates), polyoxyethylene fatty alcohols,polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fattyacid esters, glycerol polyethylene glycol ricinoleate, ethoxylatedsoybean sterols, ethoxylated castor oil or ethylene oxide (EO) andpropylene oxide (PO) block copolymers; sterol aliphatic acid estersincluding, cholesterol butyrate, cholesterol iso-butyrate, cholesterolpalmitate, cholesterol stearate, lanosterol acetate, ergosterolpalmitate, or phytosterol n-butyrate; sterol esters of sugar acidsincluding cholesterol glucuronides, lanosterol glucoronides,7-dehydrocholesterol glucoronide, ergosterol glucoronide, cholesterolgluconate, lanosterol gluconate, or ergosterol gluconate; esters ofsugar acids and alcohols including lauryl glucoronide, stearoylglucoronide, myristoyl glucoronide, lauryl gluconate, myristoylgluconate, or stearoyl gluconate; esters of sugars with aliphatic acidsincluding sucrose laurate, fructose laurate, sucrose palmitate, sucrosestearate, glucuronic acid, gluconic acid or polyuronic acid; saponinsincluding sarsasapogenin, smilagenin, hederagenin, oleanolic acid, ordigitoxigenin; glycerol or glycerol esters including glyceroltripalmitate, glycerol distearate, glycerol tristearate, glyceroldimyristate, glycerol trimyristate, glycerol dilaurate, glyceroltrilaurate, glycerol dipalmitate; long chain alcohols including n-decylalcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, or n-octadecylalcohol; 6-(5-cholesten-3β-yloxy)-1-thio-β-D-galactopyranoside;digalactosyldiglyceride;6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxy-1-thio-β-D-galactopyranoside;6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-1-thio-β-D-mannopyranoside;12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoicacid;N-[12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoyl]-2-aminopalmiticacid; N-succinyl-dioleylphosphatidylethanolamine;1,2-dioleyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol;1,3-dipalmitoyl-2-succinylglycerol;1-hexadecyl-2-palmitoylglycerophosphoethanolamine orpalmitoylhomocysteine; alkylamines or alkylammonium salts, comprising atleast one (C₁₀-C₂₀), preferably (C₁₄-C₁₈), alkyl chain, such as, forinstance, N-stearylamine, N,N′-distearylamine, N-hexadecylamine,N,N′-dihexadecylamine, N-stearylammonium chloride,N,N′-distearylammonium chloride, N-hexadecylammonium chloride,N,N′-dihexadecylammonium chloride, dimethyldioctadecylammonium bromide(DDAB), hexadecyltrimethylammonium bromide (CTAB); tertiary orquaternary ammonium salts comprising one or preferably two (C₁₀-C₂₀),preferably (C₁₄-C₁₈), acyl chain linked to the N-atom through a (C₃-C₆)alkylene bridge, such as, for instance,1,2-distearoyl-3-trimethylammonium-propane (DSTAP),1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP),1,2-oleoyl-3-trimethylammonium-propane (DOTAP),1,2-distearoyl-3-dimethylammonium-propane (DSDAP); and mixtures orcombinations thereof.

Depending on the combination of components and on the manufacturingprocess of the microbubbles, the above listed exemplary compounds may beemployed as main compound for forming the microbubble's envelope or assimple additives, thus being present only in minor amounts.

According to a preferred embodiment, at least one of the compoundsforming the microbubbles' envelope is a phospholipid, optionally inadmixture with any of the other above cited film-forming materials.According to the present description, the term phospholipid is intendedto encompass any amphiphilic phospholipid compound, the molecules ofwhich are capable of forming a stabilizing film of material (typicallyin the form of a mono-molecular layer) at the gas-water boundaryinterface in the final microbubbles suspension. Accordingly, thesematerials are also referred to in the art as “film-formingphospholipids”.

Amphiphillc phospholipid compounds typically contain at least onephosphate group and at least one, preferably two, lipophilic long-chainhydrocarbon group.

Examples of suitable phospholipids include esters of glycerol with oneor preferably two (equal or different) residues of fatty acids and withphosphoric acid, wherein the phosphoric acid residue is in turn bound toa hydrophilic group, such a, for instance, choline(phosphatidylcholines—PC), serine (phosphatidylserines—PS), glycerol(phosphatidylglycerols—PG), ethanolamine (phosphatidylethanolamines—PE),inositol (phosphatidylinositol). Esters of phospholipids with only oneresidue of fatty acid are generally referred to in the art as the “lyso”forms of the phospholipid or “lysophospholipids”. Fatty acids residuespresent in the phospholipids are in general long chain aliphatic acids,typically containing from 12 to 24 carbon atoms, preferably from 14 to22; the aliphatic chain may contain one or more unsaturations or ispreferably completely saturated. Examples of suitable fatty acidsincluded in the phospholipids are, for instance, lauric acid, myristicacid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleicacid, linoleic acid, and linolenic acid. Preferably, saturated fattyacids such as myristic acid, palmitic acid, stearic acid and arachidicacid are employed.

Further examples of phospholipid are phosphatidic acids, i.e. thediesters of glycerol-phosphoric acid with fatty acids; sphingolipidssuch as sphingomyelins, i.e. those phosphatidylcholine analogs where theresidue of glycerol diester with fatty acids is replaced by a ceramidechain; cardiolipins, i.e. the esters of 1,3-diphosphatidylglycerol witha fatty acid; glycolipids such as gangliosides GM1 (or GM2) orcerebrosides; glucolipids; sulfatides and glycosphingolipids.

As used herein, the term phospholipids include either naturallyoccurring, semisynthetic or synthetically prepared products that can beemployed either singularly or as mixtures.

Examples of naturally occurring phospholipids are natural lecithins(phosphatidylcholine (PC) derivatives) such as, typically, soya bean oregg yolk lecithins.

Examples of semisynthetic phospholipids are the partially or fullyhydrogenated derivatives of the naturally occurring lecithins. Preferredphospholipids are fatty acids di-esters of phosphatidylcholine,ethylphosphatidylcholine, phosphatidylglycerol, phosphatidic acid,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol or ofsphingomyelin.

Examples of preferred phospholipids are, for instance,dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine(DMPC), dipalmitoyl-phosphatidylcholine (DPPC),diarachidoyl-phosphatidylcholine (DAPC), distearoyl-phosphatidylcholine(DSPC), dioleoyl-phosphatidylcholine (DOPC), 1,2Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC),dipentadecanoyl-phosphatidylcholine (DPDPC),1-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC),1-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC),1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC),1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC),1-palmitoyl-2-oleylphosphatidylcholine (POPC),1-oleyl-2-palmitoyl-phosphatidylcholine (OPPC),dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts,diarachidoylphosphatidyl-glycerol (DAPG) and its alkali metal salts,dimyristoylphosphatidylglycerol (DMPG) and its alkali metal salts,dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts,distearoylphosphatidylglycerol (DSPG) and its alkali metal salts,dioleoyl-phosphatidylglycerol (DOPG) and its alkali metal salts,dimyristoyl phosphatidic acid (DMPA) and its alkali metal salts,dipalmitoyl phosphatidic acid (DPPA) and its alkali metal salts,distearoyl phosphatidic acid (DSPA), diarachidoylphosphatidic acid(DAPA) and its alkali metal salts, dimyristoyl-phosphatidylethanolamine(DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidyl-ethanolamine (DSPE), dioleylphosphatidyl-ethanolamine(DOPE), diarachidoylphosphatidylethanolamine (DAPE),dilinoleylphosphatidylethanolamine (DLPE), dimyristoylphosphatidylserine (DMPS), diarachidoyl phosphatidylserine (DAPS),dipalmitoyl phosphatidylserine (DPPS), distearoylphosphatidylserine(DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoyl sphingomyelin(DPSP), and distearoylsphingomyelin (DSSP),dilauroyl-phosphatidyllnosltol (DLPI), diarachidoylphosphatidylinositol(DAPI), dimyristoylphosphatidylinositol (DMPI),dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol(DSPI), dioleoyl-phosphatidylinositol (DOPI).

The term phospholipid further includes modified phospholipid, e.g.phospholipids where the hydrophilic group is in turn bound to anotherhydrophilic group. Examples of modified phospholipids arephosphatidylethanolamines modified with polyethylenglycol (PEG), i.e.phosphatidylethanolamines where the hydrophilic ethanolamine moiety islinked to a PEG molecule of variable molecular weight (e.g. from 300 to5000 daltons), such as DPPE-PEG (or DSPE-, DMPE- or DAPE-PEG), i.e. DPPE(or DSPE, DMPE, or DAPE) having a PEG polymer attached thereto. Forexample, DPPE-PEG2000 refers to DPPE having attached thereto a PEGpolymer having a mean average molecular weight of about 2000.

Particularly preferred phospholipids are DAPC, DSPC, DPPA, DSPA, DMPS,DPPS, DSPS and Ethyl-DSPC. Most preferred are DPPS or DSPC.

Mixtures of phospholipids can also be used, such as, for instance,mixtures of DPPE, DPPC, DSPC and/or DAPC with DSPS, DPPS, DSPA, DPPA,DSPG, DPPG, Ethyl-DSPC and/or Ethyl-DPPC.

In preferred embodiments, the phospholipid is the main component of thestabilizing envelope of microbubbles, amounting to at least 50% (w/w) ofthe total amount of components forming the envelope of the gas filledmicrobubbles. In some of the preferred embodiments, substantially thetotality of the envelope (i.e. at least 90% and up to 100% by weight)can be formed of phospholipids.

The phospholipids can conveniently be used in admixture with any of theabove listed amphiphilic compounds. Thus, for instance, lipids such ascholesterol, ergosterol, phytosterol, sitosterol, lanosterol,tocopherol, propyl gallate or ascorbyl palmitate, fatty acids such asmyristic acid, palmitic acid, stearic acid, arachidic acid andderivatives thereof or butylated hydroxytoluene and/or othernon-phospholipid compounds can optionally be added to one or more of theforegoing phospholipids in proportions ranging from zero to 50% byweight, preferably up to 25%. Particularly preferred is palmitic acid.

According to a preferred embodiment, the envelope of microbubblesforming a composition of the invention includes a compound bearing anoverall (positive or negative) net charge. Said compound can be acharged amphiphilic material, preferably a lipid or a phospholipid.

Examples of phospholipids bearing an overall negative charge arederivatives, in particular fatty acid di-ester derivatives, ofphosphatidylserine, such as DMPS, DPPS, DSPS; of phosphatidic acid, suchas DMPA, DPPA, DSPA; of phosphatidylglycerol such as DMPG, DPPG and DSPGor of phosphatidylinositol, such as DMPI, DPPI or DPPI. Also modifiedphospholipids, in particular PEG-modified phosphatidylethanolamines,such as DMPE-PEG1000, DMPE-PEG2000, DMPE-PEG3000, DMPE-PEG4000,DMPE-PEG5000, DPPE-PEG1000, DPPE-PEG2000, DPPE-PEG3000, DPPE-PEG4000,DPPE-PEG5000, DSPE-PEG1000, DSPE-PEG2000, DSPE-PEG3000, DSPE-PEG4000,DSPE-PEG5000, DAPE-PEG1000, DAPE-PEG2000, DAPE-PEG3000, DAPE-PEG4000 orDAPE-PEG5000 can be used as negatively charged molecules. Also thelyso-form of the above cited phospholipids, such aslysophosphatidylserine derivatives (e.g. lyso-DMPS, -DPPS or -DSPS),lysophosphatidic acid derivatives (e.g. lyso-DMPA, -DPPA or -DSPA) andlysophosphatidylglycerol derivatives (e.g. lyso-DMPG, -DPPG or -DSPG),can advantageously be used as negatively charged compound. Examples ofnegatively charged lipids are bile acid salts such as cholic acid salts,deoxycholic acid salts or glycocholic acid salts; and (C₁₂-C₂₄),preferably (C₁₄-C₂₂) fatty acid salts such as, for instance, palmiticacid salt, stearic acid salt, 1,2-dipalmitoyl-sn-3-succinylglycerol saltor 1,3-dipalmitoyl-2-succinylglycerol salt.

Preferably, the negatively charged compound is selected among DPPA,DPPS, DSPG, DSPE-PEG2000, DSPE-PEG5000 or mixtures thereof.

The negatively charged component is typically associated with acorresponding positive counter-ion, which can be mono- (e.g. an alkalimetal or ammonium), di- (e.g. an earth-alkali metal) or tri-valent (e.g.aluminium). Preferably the counter-ion is selected among alkali metalcations, such as Li⁺, Na⁺, or K⁺, more preferably Na⁺.

Examples of phospholipids bearing an overall positive charge arederivatives of ethyl phosphatidylcholine, in particular di-esters ofethyl phosphatidylcholine with fatty acids, such as1,2-Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC or DSEPC),1,2-Dipalmitoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DPPC or DPEPC).The negative counterion is preferably an halogen ion, in particularchlorine or bromine. Examples of positively charged lipids arealkylammonium salts with a halogen counter ion (e.g. chlorine orbromine) comprising at least one (C₁₀-C₂₀), preferably (C₁₄-C₁₈), alkylchain, such as, for instance mono or di-stearylammonium chloride, monoor di-hexadecylammonium chloride, dimethyldioctadecylammonium bromide(DDAB), hexadecyltrimethylammonium bromide (CTAB). Further examples ofpositively charged lipids are tertiary or quaternary ammonium salts witha halogen counter ion (e.g. chlorine or bromine) comprising one orpreferably two (C₁₀-C₂₀), preferably (C₁₄-C₁₈), acyl chain linked to theN-atom through a (C₃-C₆) alkylene bridge, such as, for instance,1,2-distearoyl-3-trimethylammonium-propane (DSTAP),1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP),1,2-oleoyl-3-trimethylammonium-propane (DOTAP),1,2-distearoyl-3-dimethylammonium-propane (DSDAP).

DSEPC, DPEPC and/or DSTAP are preferably employed as positively chargedcompounds in the microbubbles envelope.

The positively charged component is typically associated with acorresponding negative counter-ion, which can be mono- (e.g. halogen),di- (e.g. sulphate) or tri-valent (e.g. phosphate). Preferably thecounter-ion is selected among halogen ions, such as F⁻ (fluorine), Cl⁻(chlorine) or Br⁻ (bromine).

Mixtures of neutral and charged compounds, in particular ofphospholipids and/or lipids, can be satisfactorily employed to form themicrobubbles envelope. The amount of charged lipid or phospholipid mayvary from about 95% to about 1% by mole, with respect to the totalamount of lipid and phospholipid, preferably from 80% to 20% by mole.

Preferred mixtures of neutral phospholipids and charged lipids orphospholipids are, for instance, DPPG/DSPC, DSTAP/DAPC, DPPS/DSPC,DPPS/DAPC, DPPE/DPPG, DSPA/DAPC, DSPA/DSPC and DSPG/DSPC.

Other excipients or additives may be present either in the dryformulation of the microbubbles or may be added together with theaqueous carrier used for the reconstitution thereof, without necessarilybeing involved (or only partially involved) in the formation of thestabilizing envelope of the microbubble. These include pH regulators,osmolality adjusters, viscosity enhancers, emulsifiers, bulking agents,etc. and may be used in conventional amounts. For instance compoundslike polyoxypropylene glycol and polyoxyethylene glycol as well ascopolymers thereof can be used. Examples of viscosity enhancers orstabilizers are compounds selected from linear and cross-linked poly-and oligo-saccharides, sugars, hydrophilic polymers like polyethyleneglycol.

As the preparation of gas-filled microbubbles may involve a freezedrying or spray drying step, it may be advantageous to include in theformulation a lyophilization additive, such as an agent withcryoprotective and/or lyoprotective effect and/or a bulking agent, forexample an amino-acid such as glycine; a carbohydrate, e.g. a sugar suchas sucrose, mannitol, maltose, trehalose, glucose, lactose or acyclodextrin, or a polysaccharide such as dextran; or a polyglycol suchas polyethylene glycol.

The microbubbles of a composition according to the invention can beproduced according to any known method in the art. Typically, themanufacturing method involves the preparation of a dried powderedmaterial comprising an amphiphilic material as above indicated,preferably by lyophilization (freeze drying) of an aqueous or organicsuspension comprising said material.

For instance, as described in WO 91/15244 film-forming amphiphiliccompounds can be first converted into a lamellar form by any liposomeforming method. To this end, an aqueous solution comprising the filmforming lipids and optionally other additives (e.g. viscosity enhancers,non-film forming surfactants, electrolytes etc.) can be submitted tohigh-speed mechanical homogenisation or to sonication under acousticalor ultrasonic frequencies, and then freeze dried to form a free flowablepowder which is then stored in the presence of a gas. Optional washingsteps, as disclosed for instance in U.S. Pat. No. 5,597,549, can beperformed before freeze drying.

According to an alternative embodiment (described for instance in U.S.Pat. No. 5,597,549) a film forming compound and a hydrophilic stabiliser(e.g. polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol,glycolic acid, malic acid or maltol) can be dissolved in an organicsolvent (e.g. tertiary butanol, 2-methyl-2-butanol or C₂Cl₄F₂) and thesolution can be freeze-dried to form a dry powder.

Preferably, as disclosed in co-pending International patent applicationWO2004/069284, a phospholipid (selected among those cited above andincluding at least one of the above-identified charged phospholipids)and a lyoprotecting agent (such as those previously listed, inparticular carbohydrates, sugar alcohols, polyglycols and mixturesthereof) can be dispersed in an emulsion of water with a waterimmiscible organic solvent (e.g. branched or linear alkanes, alkenes,cyclo-alkanes, aromatic hydrocarbons, alkyl ethers, ketones, halogenatedhydrocarbons, perfluorinated hydrocarbons or mixtures thereof) underagitation. The emulsion can be obtained by submitting the aqueous mediumand the solvent in the presence of at least one phospholipid to anyappropriate emulsion-generating technique known in the art, such as, forinstance, sonication, shaking, high pressure homogenization,micromixing, membrane emulsification, high speed stirring or high shearmixing. For instance, a rotor-stator homogenizer can be employed, suchas Polytron® PT3000. The agitation speed of the rotor-stator homogenizercan be selected depending from the components of the emulsion, thevolume of the emulsion, the relative volume of organic solvent, thediameter of the vessel containing the emulsion and the desired finaldiameter of the microdroplets of solvent in the emulsion. Alternatively,a micromixing technique can be employed for emulsifying the mixture,e.g. by introducing the organic solvent into the mixer through a firstinlet (at a flow rate of e.g. 0.05-5 ml/min), and the aqueous phase asecond inlet (e.g. at a flow rate of 2-100 ml/min). The outlet of themicromixer is then connected to the vessel containing the aqueous phase,so that the aqueous phase drawn from said vessel at subsequent instantsand introduced into the micromixer contains increasing amounts ofemulsified solvent. When the whole volume of solvent has been added, theemulsion from the container can be kept under recirculation through themicromixer for a further predetermined period of time, e.g. 5-120minutes, to allow completion of the emulsion. Depending on the emulsiontechnique, the organic solvent can be introduced gradually during theemulsification step or at once before starting the emulsification step.Alternatively the aqueous medium can be gradually added to the waterimmiscible solvent during the emulsification step or at once beforestarting the emulsification step. Preferably, the phospholipid isdispersed in the aqueous medium before this latter is admixed with theorganic solvent. Alternatively, the phospholipid can be dispersed in theorganic solvent or it may be separately added the aqueous-organicmixture before or during the emulsification step. The so obtainedmicroemulsion, which contains microdroplets of solvent surrounded andstabilized by the phospholipid material (and optionally by otheramphiphilic film-forming compounds and/or additives), is thenlyophilized according to conventional techniques to obtain a lyophilizedmaterial, which is stored (e.g. in a vial in the presence of a suitablegas) and which can be reconstituted with an aqueous carrier to finallygive a gas-filled microbubbles suspension where the dimensions and sizedistribution of the microbubbles are substantially comparable with thedimensions and size distribution of the suspension of microdroplets.

A further process for preparing gas-filled microbubbles comprisesgenerating a gas microbubble dispersion by submitting an aqueous mediumcomprising a phospholipid (and optionally other amphiphilic film-formingcompounds and/or additives) to a controlled high agitation energy (e.g.by means of a rotor stator mixer) in the presence of a desired gas andsubjecting the obtained dispersion to lyophilisation to yield a driedreconstitutable product. An example of this process is given, forinstance, in WO97/29782, here enclosed by reference.

Spray drying techniques (as disclosed for instance in U.S. Pat. No.5,605,673) can also be used to obtain a dried powder, reconstitutableupon contact with physiological aqueous carrier to obtain gas-filledmicrobubbles.

The dried or lyophilised product obtained with any of the abovetechniques will generally be in the form of a powder or a cake, and canbe stored (e.g. in a vial) in contact with the desired gas. The productis readily reconstitutable in a suitable physiologically acceptableaqueous liquid carrier, which is typically injectable, to form thegas-filled microbubbles, upon gentle agitation of the suspension.Suitable physiologically acceptable liquid carriers are sterile water,aqueous solutions such as saline (which may advantageously be balancedso that the final product for injection is not hypotonic), or solutionsof one or more tonicity adjusting substances such as salts or sugars,sugar alcohols, glycols or other non-ionic polyol materials (eg.glucose, sucrose, sorbitol, mannitol, glycerol, polyethylene glycols,propylene glycols and the like).

Mean dimensions and size distribution of the final reconstitutedmicrobubbles can be in general be determined by suitably acting on theparameters of the preparation process. In general, different values ofmean size and size distribution of a final preparation can be obtainedby selecting different envelope-stabilizing phospholipids and/or (whenrequired by the process) by the selection of different organic solventsand/or different volumes thereof (relative to the volume of aqueousphase). In addition, for the specific preparation processes disclosed inWO2004/069284 or WO97/29782, a variation of the mixing speed generallyresults in a variation of the mean dimensions of the final microbubblepreparation (typically, the higher the mixing speeds, the smaller theobtained microbubbles).

Microcapsules

Gas-filled microcapsules as defined herein comprise microvesicles havinga material envelope, the thickness of which is in general much greaterthan the thickness of microbubbles stabilizing film-envelope. Dependingfrom the material forming said envelope (which can be e.g. polymeric,proteinaceous, of a water insoluble lipid or of any combinationthereof), said thickness is in general of at least 50 nm, typically ofat least 100 nm, up to few hundred nanometers (e.g. 300 nm).

Preferred examples of microcapsules are those having a stabilizingenvelope comprising a polymer, preferably a biodegradable polymer, or astabilizing envelope comprising a biodegradable water-insoluble lipid,such as, for instance those described in U.S. Pat. No. 5,711,933 andU.S. Pat. No. 6,333,021, herein incorporated by reference in theirentirety. Microcapsules having a proteinaceous envelope, i.e. made ofnatural proteins (albumin, haemoglobin) such as those described in U.S.Pat. No. 4,276,885 or EP-A-0 324 938, can also be employed

Polymers forming the envelope of the injectable microcapsules arepreferably hydrophilic, biodegradable physiologically compatiblepolymers. Examples of such polymers, which may be natural or synthetic,are substantially insoluble polysaccharides (e.g. chitosan or chitin),polycyanoacrylates, polylactides and polyglycolides and theircopolymers, copolymers of lactides and lactones such as γ-caprolactoneor δ-valerolactone, copolymers of ethyleneoxide and lactides,polyethyleneimines, polypeptides, and proteins such as gelatin,collagen, globulins or albumins. Other suitable polymers mentioned inthe above cited U.S. Pat. No. 5,711,933 include poly-(ortho)esters,polylactic and polyglycolic acid and their copolymers (e.g. DEXON®,Davis & Geck, Montreal, Canada); poly(DL-lactide-co-γ-caprolactone),poly(DL-lactide-co-δ-valerolactone),poly(DL-lactide-co-γ-butyrolactone), polyalkylcyanoacrylates;polyamides, polyhydroxybutyrate; polydioxanone; poly-β-aminoketones;polyphosphazenes; and polyanhydrides. Polyamino-acids such aspolyglutamic and polyaspartic acids can also be used, as well as theirderivatives, such as partial esters with lower alcohols or glycols.Copolymers with other amino acids such as methionine, leucine, valine,proline, glycine, alanine, etc. can also be used. Derivatives ofpolyglutamic and polyaspartic acid with controlled biodegradability(such as those described in WO87/03891, U.S. Pat. No. 4,888,398 or EP130935, all herein incorporated by reference) can also be used. Thesepolymers (and copolymers with other amino-acids) have formulae of thefollowing type: —(NH—CHA-CO)_(w)—(NH—CHX—CO)_(y)— where X designates theside chain of an amino acid residue (e.g. methyl, isopropyl, isobutyl,or benzyl); A is a group of formula —(CH₂)_(n)COOR¹R²—OCOR,—(CH₂)_(n)COO—CHR¹COOR,—(CH₂)_(n)CO(NH—CHX—CO)_(m)NH—CH(COOH)—(CH₂)_(p)COOH, or the respectiveanhydrides thereof, wherein R¹ and R² represent H or lower alkyls, and Rrepresents alkyl or aryl; or R and R¹ are connected together by asubstituted or unsubstituted linking member to provide 5- or 6-memberedrings; n, m and p are lower integers, not exceeding 5; and w and y areintegers selected for having molecular weights not below 5000.

Non-biodegradable polymers (e.g. for making microcapsules to be used inthe digestive tract) can be selected from most water-insoluble,physiologically acceptable, bioresistant polymers including polyolefins(polystyrene), acrylic resins (polyacrylates, polyacrylonitrile),polyesters (polycarbonate), polyurethanes, polyurea and theircopolymers. ABS (acryl-butadiene-styrene) is a preferred copolymer.

Biodegradable water-insoluble lipids useful for forming a microcapsulecomprise, for instance, solid water insoluble mono-, di- ortri-glycerides, fatty acids, fatty acid esters, sterols such ascholesterol, waxes and mixtures thereof. Mono-, di- and tri-glyceridesinclude mainly the mono-, di- and tri-laurin compounds as well as thecorresponding -myristin, -palmitin, -stearin, -arachidin and -beheninderivatives. Mono-, di- and tri-arachidin, -palmitin-stearin and mixedtriglycerides such as dipalmitoylmonooleyl glyceride are particularlyuseful; tripalmitin and tristearin are preferred. Fatty acids includesolid (at room temperature, about 18-25° C.) fatty acids (preferablysaturated) having 12 carbon atoms or more, including, for instance,lauric, arachidic, behenic, palmitic, stearic, sebacic, myristic,cerotinic, melissic and erucic acids and the fatty acid esters thereof.Preferably, the fatty acids and their esters are used in admixture withother glycerides.

The sterols are preferably used in admixture with the other glyceridesand or fatty acids and are selected from cholesterol, phytosterol,lanosterol, ergosterol, etc. and esters of the sterols with the abovementioned fatty acids; however, cholesterol is preferred.

Preferred biodegradable lipids are triglycerides such as tripalmitin,triarachidin, tristearin or mixtures of the above mentionedtriglycerides.

Optionally, up to 75% by weight of a biodegradable polymer, such asthose listed previously, can be admixed together with the biodegradablewater insoluble lipid forming the envelope of the microcapsule.

Advantageously, ionic polymers (i.e. polymers bearing ionic moieties intheir structure), preferably biodegradable ionic polymers, can also beused to form the stabilizing envelope of the microcapsules, thusconferring the desired overall net charge thereto. Ionic polymers can beused as main components of the stabilizing envelope or they can beadmixed in various amounts (e.g. from 2 to 80% by weight) with non ionicpolymers. Suitable ionic polymers are, for instance, polymers comprisinga quaternized nitrogen atom, such as quaternized amines or polymerscomprising an carboxylic, sulfate, sulfonate or phosphonate moieties.Examples of suitable ionic polymers include, without limitation,polyethylenimine, poly(diallyldimethylammonium chloride),poly{bis(2-chloroethyl)ether-alt-1,3-bis[3-(dimethylamino)propyl]urea}quaternized (Polyquaternium®-2), poly(4-vinylpyridinium tribromide),hydroxyethylcellulose ethoxylate quaternized (Polyquaternium®-4,poly(p-xylene tetrahydrothiophenium chloride), poly(L-lysine), chitin,diethyleneaminoethyl dextran, poly(acrylic acid), poly(methacrylicacid), poly(styrene-alt-maleic acid), poly(amino acids), alginic acid,poly(uridylic acid), hyaluronic acid, i.e. poly(β-glucuronicacid-alt-β-N-acetylglucosamide), poly(galacturonic acid), poly(vinylacetate-co-crotonic acid), DNA,poly(3,3′,4,4′-benzophenonetetracarboxylicdianhydride-co-4,4′-oxydianiline), poly(isoprene-graft-maleic acidmonomethyl ether), copolymer of glutamic acid with alkyl glutamate,heparin, poly(styrene sulfonate), sulfonated poly(Isophthalic acid),poly(vinyl sulfonate, potassium salt), poly(vinyl sulfate, potassiumsalt), chondroitin sulfate A, dextran sulfate, fucoidan, polyphosphoricacid, sodium polyphosphate, sodium polyvinylphosphonate, poly-L-lysinehydrobromide, chitosan, chitosan sulfate, sodium alginate, alginic acidand ligninsulfonate.

Conventional additives can also be incorporated into the envelope of themicrocapsules, to modify physical properties thereof, such asdispersibility, elasticity and water permeability. In particular,effective amounts of amphiphilic materials can be added to the emulsionprepared for the manufacturing of said microcapsules, in order toincrease the stability thereof. Said materials can advantageously beselected among those amphiphilic compounds, such as lipids,phospholipids and modified phospholipids, listed in the foregoing ofthis specification.

The added amphiphilic material can advantageously be a compound bearingan overall net charge. Preferred charged lipids, phospholipids andmodified phospholipids are those previously listed. Preferably theamount of charged compound, when present, is from about 2% to 40% of thetotal weight of the material forming the stabilizing envelope.

Other excipients or additives, in particular used for the preparation ofmicrocapsules, can be incorporated into the envelope such asredispersing agents or viscosity enhancers.

Biodegradable polymer-containing microcapsules can be prepared, forinstance, according to the process disclosed in U.S. Pat. No. 5,711,933,herein incorporated by reference, which comprises (a) emulsifying ahydrophobic organic phase into a water phase so as to obtain droplets ofsaid hydrophobic phase as an oil-in-water emulsion in said water phase;(b) adding to said emulsion a solution of at least one polymer in avolatile solvent insoluble in the water phase, so that said polymerforms a layer around said droplets; (c) evaporating said volatilesolvent so that the polymer deposits by interfacial precipitation aroundthe droplets which then form beads with a core of said hydrophobic phaseencapsulated by a membrane of said polymer, said beads being insuspension in said water phase; (d) removing said encapsulatedhydrophobic phase by evaporation by subjecting said suspension toreduced pressure; and (e) replacing said evaporated hydrophobic phasewith a suitable gas.

Biodegradable lipid-containing microcapsules can be prepared, forinstance, according to the process disclosed in U.S. Pat. No. 6,333,021(herein incorporated by reference), by dispersing a mixture of one ormore of the solid constituents of the microcapsule envelope dissolved inan organic solvent in a water carrier phase, so as to produce anoil-in-water emulsion. The emulsion water phase may contain an effectiveamount of amphiphilic materials which are used to stabilise theemulsion.

A certain amount of redispersing agent and/or of a cryoprotecting orlyoprotecting agent, such as those previously indicated, is then addedto the emulsion of tiny droplets of the organic solution in the waterphase, prior to freezing at a temperature below −30° C. Any convenientredispersing agent may be used; redispersing agents selected fromsugars, albumin, gelatine, polyvinyl pyrolidone (PVP), polyvinyl alcohol(PVA), polyethylene glycol (PEG) and ethyleneoxide-propyleneoxide blockcopolymer (e.g. Pluronic®, or Synperonic®) or mixtures thereof arepreferred. The redispersing agents which are added to prevent particleagglomeration are particularly useful when the microcapsules are in theform of non-coalescent, dry and instantly dispersible powders. Thefrozen emulsion is then subjected to reduced pressure to effectlyophilisation, i.e. the removal by sublimation of the organic solventfrom the droplets and of the water of the carrier phase, and thefreeze-dried product is then contacted with the desired gas.

The microcapsules can then be reconstituted by contacting the driedpowder with a suitable aqueous carrier under gentle agitation.

Biocompatible Gas

Any biocompatible gas, gas precursor or mixture thereof may be employedto fill the above microvesicles.

The gas may comprise, for example, air; nitrogen; oxygen; carbondioxide; hydrogen; nitrous oxide; a noble or inert gas such as helium,argon, xenon or krypton; a radioactive gas such as Xe¹³³ or Kr⁸¹; ahyperpolarized noble gas such as hyperpolarized helium, hyperpolarizedxenon or hyperpolarized neon; a low molecular weight hydrocarbon (e.g.containing up to 7 carbon atoms), for example an alkane such as methane,ethane, propane, butane, isobutane, pentane or isopentane, a cycloalkanesuch as cyclobutane or cyclopentane, an alkene such as propene, buteneor isobutene, or an alkyne such as acetylene; an ether; a ketone; anester; halogenated gases, preferably fluorinated gases, such as orhalogenated, fluorinated or prefluorinated low molecular weighthydrocarbons (e.g. containing up to 7 carbon atoms); or a mixture of anyof the foregoing. Where a halogenated hydrocarbon is used, preferably atleast some, more preferably all, of the halogen atoms in said compoundare fluorine atoms.

Fluorinated gases are preferred, in particular perfluorinated gases,especially in the field of ultrasound imaging. Fluorinated gases includematerials which contain at least one fluorine atom such as, for instancefluorinated hydrocarbons (organic compounds containing one or morecarbon atoms and fluorine); sulfur hexafluoride; fluorinated, preferablyperfluorinated, ketones such as perfluoroacetone; and fluorinated,preferably perfluorinated, ethers such as perfluorodiethyl ether.Preferred compounds are perfluorinated gases, such as SF₆ orperfluorocarbons (perfluorinated hydrocarbons), i.e. hydrocarbons whereall the hydrogen atoms are replaced by fluorine atoms, which are knownto form particularly stable microbubble suspensions, as disclosed, forinstance, in EP 0554 213, which is herein incorporated by reference.

The term perfluorocarbon includes saturated, unsaturated, and cyclicperfluorocarbons. Examples of biocompatible, physiologically acceptableperfluorocarbons are: perfluoroalkanes, such as perfluoromethane,perfluoroethane, perfluoropropanes, perfluorobutanes (e.g.perfluoro-n-butane, optionally in admixture with other isomers such asperfluoro-isobutane), perfluoropentanes, perfluorohexanes orperfluoroheptanes; perfluoroalkenes, such as perfluoropropene,perfluorobutenes (e.g. perfluorobut-2ene) or perfluorobutadiene;perfluoroalkynes (e.g. perfluorobut-2-yne); and perfluorocycloalkanes(e.g. perfluorocyclobutane, perfluoromethylcyclobutane,perfluorodimethylcyclobutanes, perfluorotrimethylcyclobutanes,perfluorocyclopentane, perfluoromethylcyclopentane,perfluorodimethylcyclopentanes, perfluorocyclohexane,perfluoromethylcyclohexane and perfluorocycloheptane). Preferredsaturated perfluorocarbons have the formula C_(n)F_(n+2), where n isfrom 1 to 12, preferably from 2 to 10, most preferably from 3 to 8 andeven more preferably from 3 to 6. Suitable perfluorocarbons include, forexample, CF₄, C₂F₆, C₃F₈, C₄F₈, C₄F₁₀, C₅F₁₂, C₆F₁₂, C₆F₁₄, C₇F₁₄,C₇F₁₆, C₈F₁₈, and C₉F₂₀.

Particularly preferred gases are SF₆ or perfluorocarbons selected fromCF₄, C₂F₆, C₃F₈, C₄F₈, C₄F₁₀ or mixtures thereof; SF₆, C₃F₈ or C₄F₁₀ areparticularly preferred.

It may also be advantageous to use a mixture of any of the above gasesin any ratio. For instance, the mixture may comprise a conventional gas,such as nitrogen, air or carbon dioxide and a gas forming a stablemicrobubble suspension, such as sulfur hexafluoride or a perfluorocarbonas indicated above. Examples of suitable gas mixtures can be found, forinstance, in WO 94/09829, which is herein incorporated by reference. Thefollowing combinations are particularly preferred: a mixture of gases(A) and (B) in which the gas (B) is a fluorinated gas, preferablyselected from SF₆, CF₄, C₂F₆, C₃F₆, C₃F₈, C₄F₆, C₄F₈, C₄F₁₀, C₅F₁₀,C₅F₁₂ or mixtures thereof, and (A) is selected from air, oxygen,nitrogen, carbon dioxide or mixtures thereof. The amount of gas (B) canrepresent from about 0.5% to about 95% v/v of the total mixture,preferably from about 5% to 80%.

In certain circumstances it may be desirable to include a precursor to agaseous substance (i.e. a material that is capable of being converted toa gas in vivo). Preferably the gaseous precursor and the gas derivedtherefrom are physiologically acceptable. The gaseous precursor may bepH-activated, photo-activated, temperature activated, etc. For example,certain perfluorocarbons may be used as temperature activated gaseousprecursors. These perfluorocarbons, such as perfluoropentane orperfluorohexane, have a liquid/gas phase transition temperature aboveroom temperature (or the temperature at which the agents are producedand/or stored) but below body temperature; thus, they undergo aliquid/gas phase transition and are converted to a gas within the humanbody.

For ultrasonic echography, the biocompatible gas or gas mixture ispreferably selected from air, nitrogen, carbon dioxide, helium, krypton,xenon, argon, methane, halogenated hydrocarbons (including fluorinatedgases such as perfluorocarbons and sulfur hexafluoride) or mixturesthereof. Advantageously, perfluorocarbons (in particular C₄F₁₀ or C₃F₈)or SF₆ can be used, optionally in admixture with air or nitrogen.

For the use in MRI the microvesicles will preferably contain ahyperpolarized noble gas such as hyperpolarized neon, hyperpolarizedhelium, hyperpolarized xenon, or mixtures thereof, optionally inadmixture with air, CO₂, oxygen, nitrogen, helium, xenon, or any of thehalogenated hydrocarbons as defined above.

For use in scintigraphy, the microvesicle will preferably containradioactive gases such as Xe¹³³ or Kr⁸¹ or mixtures thereof, optionallyin admixture with air, CO₂, oxygen, nitrogen, helium, kripton or any ofthe halogenated hydrocarbons as defined above.

Modified Microvesicles

Microvesicles useful for a composition according to the inventionoptionally comprises (e.g. contains or is associated to) a targetingligand, a diagnostic agent and/or a bioactive agent.

The term “targeting ligand” includes within its meaning any compound,moiety or residue having, or being capable to promote, a targetingactivity (e.g. Including a selective binding) of the microvesicles of acomposition of the invention towards any biological or pathological sitewithin a living body. Targets to which targeting ligand may beassociated include tissues such as, for instance, myocardial tissue(including myocardial cells and cardiomyocites), membranous tissues(including endothelium and epithelium), laminae, connective tissue(including interstitial tissue) or tumors; blood clots; and receptorssuch as, for instance, cell-surface receptors for peptide hormones,neurotransmitters, antigens, complement fragments, and immunoglobulinsand cytoplasmic receptors for steroid hormones.

The targeting ligand may be synthetic, semi-synthetic, ornaturally-occurring. Materials or substances which may serve astargeting ligands include, for example, but are not limited to proteins,including antibodies, antibody fragments, receptor molecules, receptorbinding molecules, glycoproteins and lectins; peptides, includingoligopeptides and polypeptides; peptidomimetics; saccharides, includingmono and polysaccharides; vitamins; steroids, steroid analogs, hormones,cofactors, bioactive agents and genetic material, including nucleosides,nucleotides and polynucleotides.

Examples of suitable targets and targeting ligands are disclosed, forinstance, in U.S. Pat. No. 6,139,819, which is herein incorporated byreference.

The targeting ligand can be a compound per se which is admixed with theother components of the microvesicle or can be a compound which is boundto an amphiphilic molecule employed for the formation of themicrovesicle.

In one preferred embodiment, the targeting ligand can be bound to anamphiphilic molecule of the microvesicle through a covalent bond. Insuch a case, the specific reactive moiety that needs to be present onthe amphiphilic molecule will depend on the particular targeting ligandto be coupled thereto. As an example, if the targeting ligand can belinked to the amphiphilic molecule through an amino group, suitablereactive moieties for the amphiphilic molecule may be isothiocyanategroups (that will form a thiourea bond), reactive esters (to form anamide bond), aldehyde groups (for the formation of an imine bond to bereduced to an alkylamine bond), etc.; if the targeting ligand can belinked to the amphiphilic molecule through a thiol group, suitablecomplementary reactive moieties for the amphiphilic molecule includehaloacetyl derivatives or maleimides (to form a thioether bond); and ifthe targeting ligand can be linked to the amphiphilic molecule through acarboxylic group, suitable reactive moieties for the amphiphilicmolecule might be amines and hydrazides (to form amide or alkylamidebonds). In order to covalently bind a desired targeting ligand, at leastpart of the amphiphilic compound forming the microvesicle shall thuscontain a suitable reactive moiety and the targeting ligand containingthe complementary functionality will be linked thereto according toknown techniques, e.g. by adding it to a dispersion comprising theamphiphilic components of the microvesicle. The amphiphilic compound canbe combined with the desired targeting ligand before preparing themicrovesicle, and the so obtained combination can be used in thepreparation process of the microvesicle. Alternatively, the targetingligand can be linked to the respective amphiphilic compound during thepreparation process of the microvesicle.

According to an alternative embodiment, the targeting ligand may also besuitably associated to the microvesicle via physical and/orelectrostatic interaction. As an example, a functional moiety having ahigh affinity and selectivity for a complementary moiety can beintroduced into the amphiphilic molecule, while the complementary moietywill be linked to the targeting ligand. For instance, an avidin (orstreptavidin) moiety (having high affinity for biotin) can be covalentlylinked to a phospholipid while the complementary biotin moiety can beincorporated into a suitable targeting ligand, e.g. a peptide or anantibody. The biotin-labelled targeting ligand will thus be associatedto the avidin-labelled phospholipid of the microvesicle by means of theavidin-biotin coupling system. Alternatively, both the phospholipid andthe targeting ligand can be provided with a biotin moiety andsubsequently coupled to each other by means of avidin (which is abifunctional component capable of bridging the two biotin moieties).Examples of biotin/avidin coupling of phospholipids and peptides arealso disclosed in the above cited U.S. Pat. No. 6,139,819.Alternatively, van der Waal's interactions, electrostatic interactionsand other association processes may associate or bind the targetingligand to the amphiphilic molecules.

According to an alternative embodiment, the targeting ligand can be acompound which is admixed with the components forming the microvesicle,to be eventually incorporated the microvesicle structure, such as, forinstance, a lipopeptide as disclosed e.g. in International patentApplications WO 98/18501 or 99/55383, both herein incorporated byreference.

Alternatively, a microvesicle can first be manufactured, which comprisesa compound having a suitable moiety capable of interacting with acorresponding complementary moiety of a targeting ligand; thereafter,the desired targeting ligand is added to the microvesicle suspension, tobind to the corresponding complementary moiety on the microvesicle.Examples of suitable specific targets to which the microvesicles can bedirected are, for instance, fibrin and the GPIIbIIIa binding receptor onactivated platelets. Fibrin and platelets are in fact generally presentin “thrombi”, i.e. coagula which may form in the blood stream and causea vascular obstruction. Suitable binding peptides are disclosed, forinstance, in the above cited U.S. Pat. No. 6,139,819. Further bindingpeptides specific for fibrin-targeting are disclosed, for instance, inInternational patent application WO 02/055544, which is hereinincorporated by reference.

Other examples of important targets include receptors in vulnerableplaques and tumor specific receptors, such as kinase domain region (KDR)and VEGF (vascular endothelial growth factor)/KDR complex. Bindingpeptides suitable for KDR or VEGF/KDR complex are disclosed, forinstance, in International Patent application WO 03/74005 and WO03/084574, both herein incorporated by reference.

The term “diagnostic agent” includes within its meaning any compound,composition or particle which may be used in connection with methods forimaging an internal region of a patient and/or diagnosing the presenceor absence of a disease in a patient. In particular, diagnostic agentsincorporated into or associated to a microvesicle in a composition ofthe invention are any compound, composition or particle which may allowimaging enhancement in connection with diagnostic techniques, including,magnetic resonance imaging, X-ray, in particular computed tomography,optical imaging, nuclear imaging or molecular imaging. Examples ofsuitable diagnostic agents are, for instance, magnetite nanoparticles,iodinated compounds, such as Iomeprol®, or paramagnetic ion complexes,such as hydrophobic gadolinium complexes.

The term “therapeutic agent” includes within its meaning any substance,composition or particle which may be used in any therapeuticapplication, such as in methods for the treatment of a disease in apatient, as well as any substance which is capable of exerting orresponsible to exert a biological effect in vitro and/or in vivo.Therapeutic agents thus include any compound or material capable ofbeing used in the treatment (including diagnosis, prevention,alleviation, pain relief or cure) of any pathological status in apatient (including malady, affliction, disease lesion or injury).Examples of therapeutic agents are drugs, pharmaceuticals, bioactiveagents, cytotoxic agents, chemotherapy agents, radiotherapeutic agents,proteins, natural or synthetic peptides, including oligopeptides andpolypeptides, vitamins, steroids and genetic material, includingnucleosides, nucleotides, oligonucleotides, polynucleotides andplasmides. Among these, drugs or pharmaceuticals are preferred.

Examples of suitable therapeutic agents include antiulcerants such ascimetidine, famotidine, ranitidine, roxatidine acetate, pantoprazole,omeprazole, lansoprazole or sucralfate; gut relaxants or prokineticssuch as propantheline bromide, camylofin (acamylophenine), dicyclomine,hyoscine butyl bromide, mebeverine, cisapride, oxybutynin, pipenzolatemethyl bromide, drotaverine, metoclopramide, clidinium bromide,isopropamide or oxyphenonium bromide; enzymes or carminatives, such aspancreatin, papain, pepsin, or amylase; hepatobiliary preparations suchas chenodeoxycholic acid, ursodeoxycholic acid, L-omithine or silymarin;antihypertensives such as clonidine, methyldopa, sodium nitroprusside,terazosin, doxazosin, (DI) hydralazine or prazosin; beta blockers suchas esmolol, celiprolol, atenolol, labetolol, propranolol, metoprolol,carvedilol, sotalol, oxyprenolol or bisoprolol; calcium channel blockerssuch as felodipine, nitrendipine, nifedipine, benidipine, verapamil,amlodipine or lacidipine; ace inhibitors such as enalapril, lisinopril,ramipril, perindopril, benazepril or captopril; angiotensin IIinhibitors such as losartan potassium; potassium channel activators,such as nicorandil; diuretics and antidiuretics such ashydrochlorothiazide, xipamide, bumetanide, amiloride, spironolactone,indapamide, triamterene, clopamide, furosemide or chlorthalidone;antianginals such as isoscorbide dinitrate, oxyfedrine, isosorbide5-mononitrate, diltiazem, erythrityl tetranitrate, trimetazidine,lidoflazine, pentaerythritol tetranitrate, glyceryl trinitrate ordilazep; coagulants such as conjugated oestrogens, diosmin, menaphthone,menadione, haemocoagulase, ethamsylate (cydanamine), rutin.flavonoids oradrenochrome monosemicarbazone; anticoagulants antithrombotics orantiplatelets such as ticlopidine, warfarin, streptokinase, phenindione,rtpa, urokinase, vasopressin, nicoumalone, heparin, low molecular weightheparins, mucopolysaccharide polysulphate or dipyridamole;antiarrhythmics such as quinidine, disopyramide, procainamide,lignocaine (lidocaine), mexiletine, amiodarone, adenosine, propafenone;drugs in cardiac failure and shock such as mephentermine, digoxin,dopamine, dobutamine or noradrenaline, vasodilators such as isoxsuprine,xanthinol nicotinate, nylidrin HCl, pentoxifylline (oxpentifylline) orcyclandelate; cardiac glycosides such as deslaneside, digitoxin, digoxinor digitalin; penicillins such as benzyl penicillin, procaine penicillin(G), benzathine penicillin (G), phenoxymethyl penicillin, penicillinG/V, bacampicillin, carbenicillin, piperacillin, ampicillin (optionallyin combination with sulbactam or probenecid), cloxacillin, oramoxycillin (optionally in combination with bromhexine, cloxacillin,carbocysteine or clavulanic acid); quinolones or fluoroquinolones suchas nalidixic acid, pefloxacin, ofloxacin, sparfloxacin, norfloxacin,ciprofloxacin, lomefloxacin, cephalosporins such as ceftizoxime,cefuroxime, cefixime, cefotaxime, cefaclor, ceftriaxone sodium,cefadroxil, cephalexin, (optionally in combination with bromhexine HClor probenecid) cefazolin, cephaloridine, ceftazidime or ceforperazone;sulphonamides such as sulphonamides, sulphamoxole, sulphadimehtoxine,cotrifamole, cotrimoxazole, trimethoprim, aminoglycosides such asgentamicin, tobramycin, neomycin, amikacin, sisomicin, kanamycin,netilmicin, polymyxins such as polymyxin-b, colistin sulphate;chloramphenicol; tetracyclines such as tetracycline, doxycycline,minocycline, demeclocycline, oxytetracycline; macrolides such aserythromycin, (optionally in combination with bromhexine),clarithromycin, vancomycin, lincomycin, azithromycin, spiramycin,roxithromycin, clindamycin, cefpirome, teicoplanin (teichomycin a2),antivirals, such as abacavir, lamivudine, acyclovir, amantadine,interferon, ribavirin, stavurdine, lamivudine or zidovudine (azt);antimalarials, such as quinine, proguanil, chloroquine, primaquine,amodiaquine, artemether, artesunate, mefloquine, pyrimethamine,arteether, mepacrine; antituberculars such as cycloserine, capreomycin,ethionamide, prothionamide, isoniazid (inh), rifampicin, rifampicinoptionally in combination with inh, isoniazide, pyrazinamide and/orethambutol; ethambutol (optionally in combination with isoniazid),streptomycin or pyrazinamide; anthelmintics & antiinfestives such aspiperazine, niclosamide, pyrantel pamoate, levamisole, diethylcarbamazine, tetramisole, albendazole, praziquantel, sodium antimonygluconate or membendazole; antileprotics such as dapsone or clofazimine;antianaerobics, antiprotozoals or antiamoebics such as tinidazole,metronidazole (optionally in combination with furazolidone ornorfloxacin), diloxanide furoate, secnidazole, hydroxyquinolones,dehydroemetine, ornidazole or furazolidone; antifungals such asfluconazole, ketoconazole, hamycin, terbinafine, econazole,amphotericin-b, nystatin, clotrimazole, griseofulvin, miconazole oritraconazole; vitamins; respiratory stimulants such as doxapramhydrochloride; antiasthmatics such as isoprenaline,salbutamol(albuterol), orciprenaline, ephedrine, terbutaline sulphate,salmeterol, aminophylline, therophylline, beclomethasone dipropionate orfluticasone propionate; antiallergics such as terfenadine, astemizole,loratadine, clemastine, dimethindene maletate, fexofenadinehydrochloride, hydroxyzine, chlorpheniramine, azatadine maleate,methdilazine, pheniramine maleate, diphenhydramine or cetrizine;skeletal muscle relaxants such as tizanidine methocarbamol,carisoprodol, valethamate, baclofen, chlormezanone or chlorzoxazone;smooth muscle relaxants such as oxyphenonium bromide, propanthelinebromide, diclomine, hyoscine buytyl bromide, mebeverine, drotaverine,clidinium bromide, isopropamide or camylofin dihydrochloride; nonsteroidal anti-inflammatory drugs such as naproxen, mefenamic acid,nimesulide, diclofenac, tenoxicam, ibuprofen (optionally in combinationwith paracetamol), meloxicam, aspirin, flurbiprofen, ketoprofen,ketoprolac, phenylbutazone, oxyphenbutazone, indomethacin or piroxicam;antineoplastic agents, such as nitrogen mustard compounds (e.g.cyclophosphamide, trofosfamide, iofosfamide, melphalan or chlorambucil),aziridines (e.g. thioepa), N-nitrosurea derivatives (e.g. carmustine,lomustine or nimustine), platinum compounds (e.g. spiroplatin,cisplatin, and carboplatin), procarbazine, dacarbazine methotrexate,adriamycin, mitomycin, ansamitocin, cytosine arabinoside, arabinosyladenine, mercaptopolylysine, vineristine, busulfan, chlorambucil,melphalan (e.g. PAM, L-PAM or phenylalanine mustard), mercaptopurine,mitotane, procarbazine hydrochloride dactinomycin (actinomycin D),daunorubicin hydrochloride, doxorubicin hydrochloride, epirubicin,plicamycin (mithramycin), mitoxantrone, bleomycin, bleomycin sulfate,aminoglutethimide, estramustine phosphate sodium, flutamide, leuprolideacetate, megestrol acetate, tamoxifen citrate, testolactone, trilostane,amsacrine (m-AMSA), asparaginase (L-asparaginase) Erwina asparaginase,etoposide (VP-16), interferon α-2a, interferon α-2b, teniposide (VM-26),vinblastine sulfate (VLB), vincristine sulfate, vindesine, paclitaxel(Taxol), methotrexate, adriamycin, arabinosyl, hydroxyurea; folic acidantagonists (e.g. aminopterin, methotraxate), antagonists of purine andpyrimidine bases (e.g. mercaptopurine, tioguanine, fluorouracil orcytarabine); narcotics, opiates or sedatives such as paregoric, codeine,morphine, opium, amobarbital, amobarbital sodium, aprobarbital,butobarbital sodium, chloral hydrate, ethchlorvynol, ethinamate,flurazepam hydrochloride, glutethimide, methotrimeprazine hydrochloride,methyprylon, midazolam hydrochloride, paraldehyde, pentobarbital,secobarbital sodium, talbutal, temazepam or triazolam; local or generalanaesthetics such as bupivacaine, chloroprocaine, etidocaine, lidocaine,mepivacaine, procaine or tetracaine, droperidol, etomidate, fentanylcitrate with droperidol, ketamine hydrochloride, methohexital sodium orthiopental; neuromuscular blockers such as atracurium mesylate,gallamine triethiodide, hexafluorenium bromide, metocurine iodide,pancuronium bromide, succinylcholine chloride, tubocurarine chloride orvecuronium bromide; or therapeutics for the hormonal system, such asgrowth hormone, melanocyte stimulating hormone, estradiol,beclomethasone dipropionate, betamethasone, cortisone acetate,dexamethasone, flunisolide, hydrocortisone, methylprednisolone,paramethasone acetate, prednisolone, prednisone, triamcinolone orfludrocortisone acetate.

The microvesicles forming a composition of the invention can also beassociated to other components such as, for instance, liposomes ormicelles, Said components can simply be admixed together with themicrovesicles or can form an assembly through a physical and/or chemicalinteraction with the stabilizing envelope of the microvesicles, e.gthrough a covalent bound, an electrostatic or ionic interaction, Van derWaals interaction, hydrophobic or hydrophylic interaction. Examples ofthese associated microvesicles compositions and of the preparationthereof are disclosed, for instance, in U.S. Pat. No. 6,258,378 and inInternational Patent Applications WO2005/063305 and WO2005/063306, allherein incorporated by reference. These components associable orassociated to the microvesicles can in turn bear any of the above listedtargeting ligands, diagnostic agents of bioactive agents, which willthus be associated to the microvesicles through said associatedcomponent. For instance, magnetite nanoparticles can be admixed with acharged amphiphilic material, such as those previously mentioned, inorder to stabilize said particles and keep them dispersed in an aqueoussolution (as disclosed for instance in U.S. Pat. No. 5,545,395, hereinincorporated by reference), in order to associate it to a microvesicle.Alternatively, gadolinium complexes can be admixed with suitablemicelle-forming compounds, for instance as disclosed in European PatentEP 804 251 (herein incorporated by reference), and the formed micellecan be associated to a microvesicle. Similarly, a therapeutic agent canbe prepared as a micellar or liposomal suspension and as such beingassociated to a microvesicle.

Preparation of Microvesicles Compositions

Compositions according to the invention can advantageously be obtainedby admixing two or more different preparations of gas-filledmicrovesicle prepared according to methods known in the art (e.g. any ofthe above mentioned preparation methods), as well as two or moreprecursors of said microvesicles preparations (e.g. as emulsions or asdried compounds).

Within the context of the present specification and claims, the term“different” when referred to at least two preparations of gas-filledmicrovesicles includes within its meaning microvesicles preparationswhich have been obtained by using at least one different processparameter for the manufacturing thereof (such as agitation speed,temperature, pressure, chemical components of the stabilizing envelopeand/or process solvent). The microvesicle preparations to be combinedwill thus differ in their chemical composition (e.g. in the compositionof the stabilizing envelope) and/or in their physical parameters (e.g.thickness of the stabilizing envelope, mean size of the microvesiclesand/or size distribution thereof), in order to obtain a desired finalcombined composition which is effectively responsive to at least twodifferent transmission frequencies. Preferably, said at least twodifferent sets of microvesicles have different D_(V50) values.

These preparations can be admixed with the desired different relativevolumetric ratios, depending e.g. from their relative composition, meandimensions and/or size distribution, in order to suitably tailor thefinal combined composition to the specific diagnostic needs.

Preferably, the respective D_(V50) values of the admixed microvesiclepreparations differ by at least 0.5 μm from each other, more preferablyby at least 1.0 μm and even more preferably by at least 1.5 μm, up toe.g. a difference 5.0 μm, depending from the specific diagnostic needs.In a preferred embodiment, at least one of said at least twomicrovesicle preparations has a relatively narrow size distribution,which allows to better control the final size-distribution of thecombined composition. In particular, said distribution is preferablydefined by a D_(V)/D_(N) ratio of from about 1.2 to 3, preferably offrom 1.2 to 2. The use of only microvesicle preparations with arelatively narrow size distribution is particularly preferred when therelevant transmission frequencies at which the contrast agent isexpected to be employed are relatively close to each other (e.g. 3 MHzor less). This may in fact help to reduce the number of microvesicleshaving an intermediate size (i.e. between the respective selected meanvalues of the two preparations) which do not contribute (or contributeto a much lesser extent) to the reflection of the echo signal.

As previously mentioned, the D_(V95) value of a combined composition ofthe invention (calculated in the range 0-10 μm) is preferably lower thanabout 8 μm, more preferably of 7 μm or lower, and even more preferablyof 6 μm or lower.

A suitable method for preparing a combined microvesicle composition isto admix the respective reconstituted suspensions of microvesicles, byadmixing the respective volumes of suspension containing the desiredvolume of gas entrapped in the microvesicles.

Alternatively, respective precursors of the microvesicle preparationscan be admixed, the mixing ratio being determined by the capacity ofeach preparation to provide a respective diagnostically effective volumeof gas-filled microvesicles. For instance, at least two separatelyobtained lyophilized preparations (e.g. obtained according to any of thepreviously described preparation methods) can be admixed in the form ofdried powders and the subsequent reconstitution of the admixedlyophilized preparations will provide the final desired combinedcomposition. Furthermore, according to a preferred embodiment, two ormore microemulsions obtainable according to the method disclosed inWO2004/069284 (or two or more suspensions obtainable, as disclosed e.g.in the above cited WO 97/29782, by mixing at high speed aphospholipid-containing suspension in the presence of a gas) can beadmixed with the desired relative volumes, then lyophilized and finallyreconstituted in a physiologically acceptable liquid carrier to give thedesired combined composition.

As an alternative to the separate preparation of microvesiclecompositions (or precursors thereof) and subsequent admixture thereof,the combined composition can advantageously be obtained by using an“all-in-one” process, whereby the combined composition is formed byapplying different process parameter to a same preparation mixture. Thismethod can be particularly useful in the case of some preparationmethods of combined microbubbles compositions.

For instance, it is possible to prepare a first emulsion according tothe process disclosed in the above cited WO2004/069284, by homogenizingwater and an organic solvent in the presence of a phospholipid at acertain speed (e.g. at 12000 rpm by means of a rotor stator mixer), toobtain a first population of microdroplets having a respective D_(V50)value. Then, an additional aliquot of solvent (the same or a differentone) and optionally of phospholipid (the same or a different one) isadded to the formed emulsion, which is then homogenized at a lower speed(e.g. 8000 rpm), thus obtaining a second population of microdropletshaving a respective D_(V50) value, in general higher than the first one,which is intimately admixed with the first one.

Similarly, when a micromixing process is used, a first emulsion (with arespective D_(V50) value) can be prepared by circulating the emulsion ata predetermined recirculation rate with a predetermined relative volumeof solvent; then the recirculation rate is lowered, by adding anadditional (equal or different) volume of an equal or different solvent,thus obtaining a second “integrated” emulsion having a higher value ofD_(V50). This process can be performed either discontinuously (i.e. bystopping the first homogenization, resetting the process parameters andperforming the second homogenization) or in a continuous manner, bychanging the homogenization parameters without stopping the processwhile adding the additional solvent.

In addition, particularly in the case of the micromixing process, theprocess parameters can be gradually modified (e.g. the recirculationrate can be stepwise decreased from 20 to 10 ml/min in 20 minutes, witha variation of 0.5 ml/min each minute) thus obtaining a finalcomposition formed by the combination of a relatively large number ofmicrobubble preparations having different mean sizes. Instead of astepwise variation, also a continuous variation of the recirculationrate can be used; In this case, a corresponding substantially infinitenumber of intimately admixed microbubble preparations having differentmean sizes will be obtained.

Similarly to the above emulsion process, also other preparation methodscan be suitably modified to result in an “all-in-one” preparation methodof a combined composition of the invention. Thus, for instance, a firstset of gas-filled microvesicles can be prepared by submitting an aqueousmedium comprising a phospholipid (and optionally other amphiphilicfilm-forming compounds and/or additives) to a first controlled agitationenergy (e.g. by means of a rotor stator mixer) in the presence of adesired gas; then, the same suspension (with the optional addition ofthe same or another phospholipid) is subjected to a second agitationenergy, lower than the first one, to obtain a second set of largermicrobubbles.

The precursors of the combined compositions obtained according to any ofthe above “all-in-one” methods can then be lyophilized according toconventional techniques to obtain a dried powder, as previouslyillustrated; the final combined composition is then obtained uponreconstitution of the lyophilized residue.

In any case, i.e. whether the combined composition is obtained by mixingtwo separately prepared microvesicles preparations or whether it isobtained according to any of the above “all-in-one” processes, the finalcombined composition will show a peculiar size distribution patternderiving from the combination of the different microvesicle preparationsforming the combined composition, regardless of how these preparationshave been admixed.

The size distribution of the microvesicles in the so obtained combinedcomposition will thus be suitable for conferring the contrast agent withan effective response at different selected working frequencies.

The present combined composition is in particular suitable for beingused at rather different transmission frequencies while showing aremarkably good image enhancement at said selected frequencies.Advantageously, the present invention allows to prepare a“multi-purpose” ultrasound contrast agent which is able of beingeffectively employed in a relatively wide range of frequencies. The sizedistribution of the composition can for instance be tailored toeffectively work at two or more different ultrasound frequencies emittedby currently employed ultrasound probes, typically from 1.5 to 15 MHz,preferably 1.5 to 10 MHz. Also lower frequencies can be contemplatedsuch as down to 0.5 MHz (e.g. for particular cardiac applications), aswell as higher frequencies, e.g. up to about 80 MHz for other specificapplications (such as intravascular ultrasound imaging).

Recent ultrasound contrast-imaging methods exploit the nonlinearscattering characteristics of gas-filled microvesicles as an ultrasoundcontrast agent (UCA). From the literature (e.g. Eatock et al., Journalof the Acoustical Society of America, vol.77(5), pp1692-1701, 1985) itis known that nonlinear scattering is significant only for a populationof microvesicles which are smaller than, or close to, resonance size,and mainly for those microvesicles that are half the resonance size.“Half the resonance size” is the size of a microvesicle with a resonancefrequency that equals twice the center frequency of the transmittedultrasound wave.

When imaging a volume containing an ultrasound contrast agent based ongas-filled microvesicles, the detectability of the microvesicles echoesagainst tissue echoes is enhanced by the level of nonlinear scatteringby the microvesicles, and decreased by the attenuation caused by themicrovesicles located between the probe and the region of interest.Attenuation along the transmit path reduces the ultrasound-energyavailable for generating nonlinear response of gas-filled microvesicles;attenuation along the receive path removes echo-energy able to reach theultrasound probe.

In the case of a suspension comprising a wide range of microvesiclesizes, at a specific transmit frequency, the microvesicles larger thanresonance size mainly contribute to transmit-receive attenuation,without contributing in an efficient way to the nonlinear (e.g. 2^(nd)harmonic) echo signals. Conversely, at said transmit frequency, theoverall acoustic response for nonlinear imaging may benefit from the useof a narrow distribution of microvesicle sizes, calibrated with a meansize close to the resonance size or smaller, preferably calibrated witha mean size between resonance size and half resonance size. For aselected frequency of transmission it is thus possible to define adistribution of microvesicle sizes having an optimal overall acousticresponse to said frequency (i.e. a peak of nonlinear echographicresponse at said selected transmission frequency), in particular with ahigh 2^(nd) harmonic scattering and low attenuation.

An example of a suitable parameter for defining a size distributionhaving an optimal overall acoustic response is the “second harmonicscattering-to attenuation ratio” or the “STAR_(H)”. The STAR_(H), andsubsequently the corresponding size distribution, can be calculated, forinstance, according to the method schematically illustrated in FIG. 3.According to said method, a response of a microvesicle composition to aultrasound wave with a selected fundamental frequency f1 (Resp. f1) isfirst simulated as a function of microvesicle size with models known inthe art, such as, for instance, the one described by Morgan et al., IEEETrans. Ultrason. Ferr. Freq. Control, vol. 47(6), pp. 1494, 2000, whichtake into account, among other parameters, also the visco-elasticparameters and the thickness of the microvesicles' stabilizing envelope(which are generally determined by its chemical composition). This firstsimulation is then used to calculate the corresponding non-linearresponse of microvesicles at a respective 2^(nd) harmonic frequency f2(non-L resp. f2). Next, the attenuation at the fundamental frequency f1(Atten. f1), due to propagation of the ultrasound wave from thetransducer to a region of Interest through a volume of microvesicles(forward propagation) is calculated as a function of microvesicle size.Finally, the attenuation at the 2^(nd) harmonic frequency f2 (Atten.f2), due to propagation of the ultrasound wave from a region of interestback to the transducer through a volume of microvesicles (backwardpropagation) is calculated as a function of microvesicle size. Theattenuation at the fundamental frequency f1 can be calculated with thesame model mentioned above or with other models such as, for instance,the one described by Gorce et al., Invest. Radiol., vol.35(11), pp661,2000. The attenuation at a 2^(nd) harmonic frequency f2, can forinstance be calculated with the same model described by Gorce et al.

Irrespective of the calculation method being used, the calculated valuesof non-L resp f2, of Atten. f1 and of Atten. f2 are combined together(e.g. with the operational modalities illustrated in FIG. 3) tocalculate the STAR_(H) as a function of microvesicle size. Based on saidSTAR_(H), a size distribution of microvesicles can be constructed bybest fit procedures having an optimal acoustic response (STAR_(H)) atthe selected frequency. For instance, FIG. 4 shows the result of theprocedure described in FIG. 3, calculated for phospholipid-stabilizedmicrobubbles, illustrating a simulated volume size distribution havingan optimal acoustic response (i.e. a peak of nonlinear echographicresponse) at frequencies around 2 MHz (solid line) and around 6 MHz(dashed line), respectively.

These or other simulations may thus allow to estimate with sufficientlygood approximation an optimal size distribution having a peak ofnonlinear echographic response at a selected transmission frequency;these results may then be used to specifically tailor a combinedcomposition of the invention effectively responsive to a selected set oftransmission frequencies. However, also in the absence of the knowledgeof the final transmission frequencies which will be used for a specificdiagnostic application, based on the teachings of the presentspecification, a generic “multi-responsive” combined composition cannevertheless easily be prepared by admixing at least two different setsof microvesicles having relatively different median (D_(V50)) sizevalues.

The following are some examples of experimental phospholipid-basedgas-filled microbubbles preparations, characterized by their respectivevalues of D_(V50), D/D_(N) and corresponding peak of non-linearechographic response (peak),:

-   Prep. 1: D_(V50)=1.7, D_(V)/D_(N)=1.4, peak≈6 MHz;-   Prep. 2: D_(V50)=1.8, D_(V)/D_(N)=1.5, peak≈6 MHz;-   Prep. 3: D_(V50)=2.5, D_(V)/D_(N)=1.8, peak≈3.5 MHz;-   Prep. 4: D_(V50)=2.9, D_(V)/D_(N)=1.85, peak≈3 MHz;-   Prep. 5: D_(V50)=3.6, D_(V)/D_(N)=2.1, peak≈2 MHz;-   Prep. 6: D_(V50)=4.1, D_(V)/D_(N)=2.2, peak≈1.5 MHz.

The above preparations can thus be admixed to obtain combinedcompositions effectively responsive to different transmissionfrequencies. The following are illustrative examples of combinedcompositions obtainable by admixing respective relative volumes of gas(RVG) of said microbubbles preparations:

Comp. 1: Prep.4/Prep.2, RVG=27/73;

Comp. 2: Prep.4/Prep.2, RVG=43/57;

Comp. 3: Prep.5/Prep.1, RVG=53/47;

Comp. 4: Prep.5/Prep.1, RVG=37/63;

Comp. 5: Prep.6/Prep.3/Prep.1, RVG=30/35/35;

Comp. 6: Prep.6/Prep.3/Prep.1, RVG=25/35/40.

Further to the difference in the respective mean size, other parameterscan also be varied to provide a combined composition being effectivelyresponsive to at least two different frequencies, such as, for instance,the thickness and the visco-elastic properties (and inherently thechemical composition) of the stabilizing envelope. For instance, apreparation of microbubbles (responsive to a first transmissionfrequency) can be admixed with a preparation of microcapsules(responsive to a second transmission frequency). According to apreferred embodiment, the microvesicles admixed to form the combinedcomposition of the invention are however substantially of the same type,i.e. they are either microbubbles or microcapsules. More preferably, themicrovesicles forming the combined composition are gas-filledphospholipid-stabilized microbubbles.

In general, the single preparations forming a combined compositionaccording to the invention can differ in further chemical, biologicaland/or physical parameters such as, for instance, their resistance toacoustic pressure, their half life in blood after intravenousadministration, their capacity of targeting or acting on a specifictissue, organ or cell and/or the possible inclusion of a diagnosticand/or of bioactive agent therein.

A combined composition according to the invention is preferably storedin dried powdered form and as such can advantageously be packaged in atwo component diagnostic and/or therapeutic kit. The kit preferablycomprises a first container, containing the lyophilized composition incontact with a selected microvesicle-forming gas and a second container,containing a physiologically acceptable aqueous carrier. Examples ofsuitable carriers are water, typically sterile, pyrogen free water (toprevent as much as possible contamination in the intermediatelyophilized product), aqueous solutions such as saline (which mayadvantageously be balanced so that the final product for injection isnot hypotonic), or aqueous solutions of one or more tonicity adjustingsubstances such as salts or sugars, sugar alcohols, glycols or othernon-ionic polyol materials (eg. glucose, sucrose, sorbitol, mannitol,glycerol, polyethylene glycols, propylene glycols and the like). Saidtwo component kit can include two separate containers or a dual-chambercontainer. In the former case the container is preferably a conventionalseptum-sealed vial, wherein the vial containing the lyophilized residueis sealed with a septum through which the carrier liquid may be injectedusing an optionally prefilled syringe. In such a case the syringe usedas the container of the second component is also used then for injectingthe contrast agent. In the latter case, the dual-chamber container ispreferably a dual-chamber syringe and once the lyophilisate has beenreconstituted and then suitably mixed or gently shaken, the containercan be used directly for injecting the contrast agent.

The contrast agents of the present invention may be used in a variety ofdiagnostic and/or therapeutic imaging techniques, including inparticular ultrasound and Magnetic Resonance. The term therapeuticimaging includes within its meaning any method for the treatment of adisease in a patient which comprises the use of a contrast imaging agent(e.g. for the delivery of a bioactive compound to a selected targetedsite or tissue) and which is capable of exerting or responsible to exerta biological effect in vitro and/or in vivo. Possible other diagnosticimaging applications include scintigraphy, light imaging, and X-rayimaging, including X-ray phase contrast imaging. A variety of imagingtechniques may be employed in ultrasound applications, for exampleincluding fundamental and harmonic B-mode imaging, pulse or phaseinversion imaging and fundamental and harmonic Doppler imaging; ifdesired three-dimensional imaging techniques may be used. Microvesiclesaccording to the invention can typically be administered in aconcentration of from about 0.01 to about 1.0 μl of gas per kg ofpatient, depending e.g. from their respective composition, the tissue ororgan to be imaged and/or the chosen imaging technique. This generalconcentration range can of course vary depending from specific imagingapplications, e.g. when signals can be observed at very low doses suchas in color Doppler or power pulse inversion.

The following examples will illustrate the invention more in detail.

EXAMPLES

In the following examples, the size distributions, volume concentrationsand number of the microbubbles (after lyophilisation and reconstitutionwith an aqueous phase) are determined by using a Coulter Counter Mark IIapparatus fitted with a 30 μm aperture with a measuring range of 0.7 to20 μm.

The D_(V95) values calculated for the microvesicles compositions of theexamples are determined considering only the population of microvesiclesup to a diameter of 10 μm.

The value of the Bowley Skewness (BS) is calculated according to theequation previously reported, taking into consideration only thepopulation of microvesicles up to a diameter of 8 μm.

Example 1

A first emulsion (E1a) is obtained according to the following procedure:20 mg of dipalmitoylphosphatidylserine (DPPS) are added to 20 ml of an10% (w/w) mannitol solution in water. The suspension is heated at 65° C.for 15 minutes and then cooled to room temperature (22° C.).Perfluoroheptane (8% v/v) is added to this aqueous phase and emulsifiedin a beaker of about 4 cm diameter by using a high speed homogenizer(Polytron T3000, probe diameter of 3 cm) for 1 minute at 8500 rpm.

A second emulsion (E1b) is obtained by using the above procedure exceptthat high speed homogenization is performed at 12000 rpm for 1 minute.Both emulsions are heated at 75° C. for 1.5 hours, cooled to roomtemperature and centrifuged (10 min, 800-1200 rpm, Sigma centrifuge3K10¹⁰) to eliminate phospholipids in excess. The separatedmicrodroplets are recovered and re-suspended in the same initial volumeof 10% mannitol.

The two emulsions are then admixed in different volume ratios, to obtainthree combined emulsions CE1A, CE1B and CE1C (see table 1).

TABLE 1 Combined emulsion Emulsion 1a (ml) Emulsion 1b (ml) CE1A 1 4CE1B 2 4 CE1C 3 4

Each emulsion (the two single and the three combined ones) is thenfrozen separately at −45° C. for 5 minutes in a respective 100 mlround-bottomed vessel and then lyophilized at room temperature at apressure of 0.2 mbar in a Christ-Alpha 2-4 freeze-drier.

Each obtained cake is exposed to an atmosphere containing a mixture ofperfluoro-n-butane and nitrogen (35/65 v/v) and then dispersed by gentlehand shaking in twice the initial volume of water, to obtain respectivemicrobubble suspensions M1a, M1b, CM1A, CM1B and CM1C. The microbubblesuspensions obtained after reconstitution with distilled water areanalyzed using a Coulter counter. The size distributions of themicrobubbles suspension obtained from the corresponding emulsions areshown in FIG. 5a-5c (solid thin line for M1a, dashed line for M1b andsolid thick line for each combined composition in the respectivefigures, i.e. CM1A in FIG. 5a , CM1B in FIG. 5b and CM1C in FIG. 5c ).

As shown in these graphs, microbubble preparations M1a and M1b (obtainedfrom emulsions E1a and E1b) show respective D_(V50) values of about 2.77μm and 1.64 μm (with respective peaks of nonlinear echographic responseat about 3 MHz and about 6 MHz), while combined preparations CM1A-CM1Cshow corresponding intermediate size distributions. From these figures,the unusual pattern of the size distributions of combined preparationscan be observed, in particular a plateau extending from about 1.5 μm toabout 3.5 μm in the case of combined preparation CM1C.

The respective BS and D_(V95) values calculated for the combinedcompositions were as follows:

-   CM1A : BS=0.20; D_(V95)=4.2-   CM1B : BS=0.19; D_(V95)=4.6-   CM1C : BS=0.19; D_(V95)=4.8

Example 2

A first suspension (S2a) is prepared by adding 200 mg of DPPS to 100 mlof water containing 5.4% (w/w) of a mixture of propylene glycol andglycerol (3:10 w/w). The resulting mixtures is shaken, heated to 80° C.for five minutes, allowed to cool to room temperature and thenintroduced in a doubled-walled reactor connected to a water bath tomaintain the temperature. The reactor is connected to an in-line rotorstator mixing system (Megatron MT40-Kinematica). Perfluoro-n-butane gas(F2 Chemicals, Preston Lancashire UK) is introduced in the liquid streambetween the reactor and the mixing system via a Y-shaped connection. Thesolution is homogenised at 25000 rpm for three minutes at roomtemperature. The resulting microbubble suspension is transferred into a100 ml syringe and after overnight decantation, the lower phase isremoved and replaced by 10% maltose solution in water.

A second suspension S2b is obtained according to the above procedurewith the only difference that the solution is homogenised for threeminutes at 17000 rpm at a temperature of 0-5° C.

Aliquots of the two suspensions are admixed in different relativeratios, to obtain three combined microbubble preparations CS2A, CS2B andCS2C, as illustrated in table 2.

TABLE 2 Combined Suspension Suspension Suspension S2a (ml) S2b (ml) CS2A20 20 CS2B 24 15 CS2C 30 10

1 ml of each preparation is introduced into a respective 10 mlflat-bottomed vial. The vials are cooled at −45° C. for 1 hour,freeze-dried (Freeze dryer Christ Epsilon 2-12DS—Main drying: −5° C./0.1mBar/5 h—Final drying:

25° C./0.1 mBar/10 h), stoppered in an atmosphere of perfluoro-n-butaneand sealed.

In order to obtain the respective final microbubble preparations M2a(from S2a), M2b (from S2b), CM2A (from CS2A), CM2B (from CS2B) and CM2C(from CS2C), water (5 ml) is added to each vial through the septum andthe vials are gently mixed. Microbubbles size distributions are measuredusing a Coulter counter as in example 1.

Preparations M2a and M2b show respective values of D_(V50) of about 1.64and 2.81 μm, with respective peaks of nonlinear echographic response ofabout 6 MHz and about 3 MHz.

FIGS. 6a-6c show (solid thick line) the size distribution of eachrespective combined composition CM2A (FIG. 6a ), CM2B (FIG. 6b ) andCM2C (FIG. 6c ), compared with the size distribution of the two singlepreparations M2a and M2b (solid thin line and dashed line,respectively). Also in this case, a particularly unusual(trapezoidal-like) size-distribution pattern can be observed for thecombined preparations of FIGS. 6a-6c , in particular in the case ofpreparation 6 a showing a substantially flat portion. The respective BSand D_(V95) values calculated for the combined compositions were asfollows:

-   CM2A : BS=0.22; D_(V95)=5.7-   CM2B : BS=0.32; D_(V95)=5.3-   CM2C : BS=0.31; D_(V95)=4.8

Example 3

20 mg of DPPS are added to 20 ml of a 10% (w/w) mannitol solution inwater. The suspension is heated at 65° C. for 15 minutes and then cooledto room temperature (22° C.). Perfluoroheptane (0.6 ml-2.9% v/v) isadded to the aqueous suspension and emulsified in a beaker of about 4 cmdiameter by using a high speed homogenizer (Polytron T3000, probediameter of 3 cm) during 1 minute at 8500 rpm, to obtain a firstemulsion (E3a).

A second emulsion (E3b) is obtained by the same procedure except thatperfluoroheptane (1 ml-4.8%) is added to the aqueous phase andemulsified at 12000 rpm for 1 minute.

A third emulsion (CE3A) is obtained by the same procedure except that 1ml of perfluoroheptane is first added to the aqueous suspension andemulsified in the aqueous phase at 12000 rpm for 1 minute; thenagitation is stopped, further 0.6 ml of perfluoroheptane are added tothe emulsion and the mixture is emulsified at a mixing speed of 8500 rpmfor an additional minute.

A fourth emulsion (CE3B) is obtained by the same procedure except that0.8 ml of perfluoroheptane are first added to the aqueous suspension andemulsified at 12000 rpm for 1 minute; then agitation is stopped, further0.8 ml of perfluoroheptane are added to the emulsion and the mixture isemulsified at a mixing speed of 8500 rpm for an additional minute.

Each obtained emulsion is heated at 75° C. for 1.5 hours, cooled to roomtemperature and then centrifuged (10 min, 1200 rpm, Sigma centrifuge3K10¹⁰) to eliminate phospholipids in excess. The separatedmicrodroplets are recovered and re-suspended in the same initial volumeof 10% mannitol.

10 ml of each of the four emulsions are then frozen separately at −45°C. for 5 min in respective 100 ml round-bottomed vessels and thenlyophilized at room temperature at a pressure of 0.2 mbar in aChrist-Alpha 2-4 freeze-drier.

Each cake is exposed to an atmosphere containing aperfluoro-n-butane/nitrogen (35/65 v/v) gas mixture and then dispersedby gentle hand shaking in twice the initial volume of water. Themicrobubble suspensions obtained after reconstitution with distilledwater are analyzed using a Coulter counter.

FIG. 7 shows the size distributions of microbubble preparations M3a(from E3a) and M3b (from E3b) in dashed thick and thin lines,respectively, and of combined compositions CM3A (from CE3A) and CM3B(from CE3B) in solid thick and thin lines, respectively.

Preparations M3a and M3b show respective values of D_(V50) of about 2.53and 1.58 μm, with respective peaks of nonlinear echographic response ofabout 3.5 MHz and about 6 MHz.

The respective BS and D_(V95) values calculated for the combinedcompositions were as follows:

-   CM3A : BS=0.24; D_(V95)=3.7-   CM3B : BS=0.24; D_(V95)=4.6

Example 4

A first emulsion (E4a) is obtained according to the following procedure:

Distearoylphosphatidylcholine (DSPC) and dipalmitoylphosphatidylserine(DPPS) are introduced at 70° C. In 40 ml of a 10% aqueous solution ofmannitol, at a concentration of 0.5 mg/ml each. After cooling to roomtemperature, this suspension is recirculated in a micromixer(Interdigital Micro-mixer, Institut für Microtechnik Mainz GmbH,Germany) at a flow rate of 20 ml/min. Cyclooctane (3.2 ml) is theninjected through the second channel at a rate of 0.2 ml/min. Theresulting emulsion is recirculated in the micromixer for 20 min.

A second emulsion (E4b) is obtained by using the above procedure exceptthat recirculation flow rate is 10 ml/min.

Both resulting emulsions are separately heated (120° C., 30 min) andthen cooled to room temperature.

Aliquots of the two emulsions are then admixed in volume ratios of 4/1or of 4/3, to obtain respective combined emulsions CE4A and CE4B.

The single and combined emulsions are finally distributed in DIN 8Rvials in 1 ml aliquots and lyophilised (Telstar Lyobeta-35freeze-drier). At the end of the lyophilization, aperfluorobutane/nitrogen mixture (35/65 v/v) is introduced in thelyophilizer and the vials are stoppered.

Upon reconstitution with distilled water, respective microbubblepreparations M4a (from E4a), M4b (from E4b), CM4A (from CE4A) and CM4B(from CE4B) are obtained.

Preparations M4a and M4b show respective values of D_(V50) of about 1.9and 2.7 μm, with respective peaks of nonlinear echographic response ofabout 6 MHz and about 3.5 MHz. The following BS and D_(V95) values weremeasured for the combined compositions:

-   CM4A : BS=0.20; D_(V95)=4.6 μm-   CM4B : BS=0.17; D_(V95)=6.8 μm

FIG. 8 shows the size distributions of microbubble preparation CM4B(thick solid line), compared with those of M4a (thin solid line) and M4b(dashed line).

Example 5

Distearoylphosphatidylcholine (DSPC) and dipalmitoylphosphatidylserine(DPPS) are introduced at 70° C. in 40 ml of a 10% aqueous solution ofmannitol, at a concentration of 0.5 mg/ml each. After cooling to roomtemperature, this suspension is recirculated in a micromixer(Interdigital Micro-mixer, Institut für Microtechnik Mainz GmbH,Germany) at a flow rate of 20 ml/min. Cyclooctane (1.6 ml) is theninjected through the second channel at a rate of 0.2 ml/min. Theresulting emulsion is recirculated in the micromixer for 20 min. Therecirculation rate is then reduced to 10 ml/min and a second amount ofcyclooctane (1.6 ml) is introduced in the second channel of themicromixer at a flow rate of 0.2 ml/min. The emulsion is recirculated ata flow rate of 10 ml/min during 20 min. The resulting emulsion iscollected, heated (120° C., 30 min), distributed in DIN 8R vials in 1 mlaliquots and lyophilised (Telstar Lyobeta-35 freeze-drier). At the endof the lyophilization, a perfluorobutane/nitrogen mixture (35/65 v/v) isintroduced in the lyophilizer and the vials are stoppered.

The microbubble suspensions obtained after reconstitution with distilledwater are analyzed using a Coulter counter.

The size distribution of the obtained microbubble preparation (BS=0.19,D_(V95)=4.9 μm) is shown in FIG. 9 shows (solid thick line),illustratively compared with preparations M4a and M4b of example 4 (thinsolid line and dashed line, respectively).

Example 6

The echographic response of an ultrasound contrast agent (UCA) accordingto the invention (composition CM1B prepared according to example 1) iscompared with the response of a commercial UCA, Sonovue® (BraccoInternational B.V.) at two different transmission frequencies, 2 MHz and10 MHz. FIG. 13 shows the size distribution of CM1B (dashed line)compared with the size distribution of Sonovue® (solid line). Table 3shows the Dv95 values and the BS of the two UCA.

TABLE 3 D_(V95) [μm] BS Sonovue 9.63 −0.05 CM1B 4.58 0.19

Different suspensions of the two UCA are prepared by adding differentvolumes of UCA to 800 mL of 0.9% NaCl, to obtain various concentrationsof the two UCA to be tested at the two different transmissionfrequencies.

For a first experiment (2 MHz transmission frequency), a setupschematically represented in FIG. 11 is adopted. This setup comprises abeaker 111 in which a tissue-mimicking phantom 112 (Model #528, ATSLaboratories Inc., Bridgeport, Conn.) is placed, immersed in arespective UCA suspension 113. A region of interest (ROI) 114 is definedat a distance A of about 7.5 cm from the transducer 115, and is used formeasuring 2^(nd) harmonic scattering including a long propagating paththrough the UCA (simulating imaging through the left ventricle forexample). The data from this ROI can therefore be interpreted as ameasure for the 2^(nd) harmonic scattering-to-attenuation ratio. A Megasultrasound system (Esaote, Florence, Italy), not shown, with a PA230Ephased array probe is used at a transmission frequency of 2 MHz. Thefocal distance is 6.5 cm and the depth is set to 25 cm to minimizereverberations within the UCA containing cavity. The mechanical index(MI), calculated from calibration measurements in water is 0.11. Thecorresponding value including a thickness B of about 3.5 cm oftissue-mimicking phantom material is 0.071. The Megas ultrasound systemis interfaced to a Femmina platform (Scabia et al. “Hardware andsoftware platform for processing and visualization of echographicradio-frequency signals”; IEEE Trans. Ultra. Ferr. Freq. Contr., 49(10),1444-1452, 2002) through an optical fiber link for the collection ofradio frequency (RF) data. The RF data are stored on a PC and processedoff-line with Matlab (version 6.5; The Mathworks Inc., Natick, Mass.).Mean power spectral density is calculated around the 2^(nd) harmonicfrequency (4 MHz) with a 0.6 MHz bandwidth, in the ROI. During themeasurements, the UCA is kept under agitation by continuous stirring, bymeans of magnetic stirrer 106. Between acquisitions, the transducer isdisconnected from the Megas to prevent overexposure and possibledestruction of the bubbles. Measurements without UCA are performed toquantify background noise.

For a second experiment, the Megas ultrasound system is replaced by aSequoia ultrasound system (Siemens Medical Systems) with a 15L8-S lineararray probe at 10 MHz in Contrast Pulse Sequencing (CPS) mode. The samesetup shown in FIG. 11 is used, with the only difference that the tissuemimicking phantom is removed and the ROI is shifted to a distance ofabout 2 cm from the transducer. The focal distance is 2.5 cm and thedepth is set to 8 cm to minimize reverberations within the UCAcontaining cavity. The MI, calculated from calibration measurements inwater is 0.13. Video images are stored on digital video (DV) cassetteand analyzed off-line using a videodensitometry program allowinggrayscale linearization to obtain a signal which is proportional toagent concentration. Between the acquisitions, the Sequoia system is setinto freeze mode to prevent overexposure and possible destruction of thebubbles. Measurements without UCA are performed to quantify backgroundnoise.

FIGS. 12a and 12b show the echo power, i.e. 2^(nd)harmonic-scattering-to-attenuation ratio as a function of agentconcentration measured with the Megas (2 MHz) and Sequoia (10 MHz)systems, respectively. The dashed lines show the results observed forSonovue® and the solid lines show the results for the CM1B formulation.As shown in these figures, both agents show very similar performances at2 MHz with a slightly improved performance for the CM1B at higherconcentrations (>0.0001 μl/ml). At 10 MHz, the CM1B formulation shows amarked improvement over Sonovue® for the whole range of concentrationsused. Particularly at the low and medium concentrations (<0.0002 μl/ml),the 2^(nd) harmonic scattering-to-fundamental ratio of the CM1Bformulation is almost 6 dB higher (almost 4×) than the one of Sonovue®.

The invention claimed is:
 1. A method of manufacturing a contrast agentcomprising gas-filled microvesicles, which comprises admixing at leasttwo different preparations of gas-filled microvesicles or precursorsthereof having respective peaks of non-linear echographic responsediffering by at least 2 MHz to each other wherein: a) said gas-filledmicrovesicles are microbubbles stabilized by a film layer of anamphiphilic material; b) the volume size distribution of said gas-filledmicrovesicles, determined on a population of microvesicles with adiameter up to 8 μm, has a Bowley skewness of 0.16 or higher; and c)said contrast agent has an effective echographic response to at leasttwo transmission frequencies differing by at least 2 MHz to each other.2. A method according to claim 1 wherein said precursors are in the formof a dried powder forming said microvesicle preparation uponreconstitution in a pharmaceutically acceptable liquid carrier.
 3. Amethod according to claim 2 wherein the at least two differentpreparations of gas-filled microvesicles or precursors thereof aredirectly obtained as a combined preparation by applying differentprocess parameter to a same preparation mixture.
 4. The method accordingto claim 1 wherein said Bowley skewness is from 0.18 to 0.40.
 5. Themethod according to claim 1 wherein said Bowley skewness is from 0.20 to0.40.
 6. The method according to claim 1 wherein at least 95% of thetotal volume of gas contained in said microvesicles, determined on apopulation of microvesicles with a diameter up to 10 μm, is contained inmicrovesicles having a diameter of 8 μm or less.
 7. The method accordingto claim 1 wherein said amphiphilic material is a phospholipid.
 8. Themethod according to claim 1 wherein the contrast agent further comprisesa physiologically acceptable aqueous carrier.
 9. The method according toclaim 1 wherein said gas-filled microvesicles are in the form of a driedpowder reconstitutable upon contact with a physiologically acceptableaqueous carrier.
 10. The method according to claim 1, wherein saidgas-filled microvesicles comprise a targeting ligand, a diagnosticagent, a bioactive agent or any combination thereof.
 11. The methodaccording to claim 1, wherein said contrast agent has an effectiveechographic response to a first transmission frequency of from 1.5 to 3MHz and to a second transmission frequency of from 5 to 10 MHz.
 12. Themethod according to claim 11, wherein said first transmission frequencyis 2 MHz and said second transmission frequency is 10 MHz.
 13. A methodaccording to claim 1 wherein said precursors are microemulsions obtainedby dispersing a phospholipid in an emulsion of water with a waterimmiscible organic solvent, said emulsion forming a microvesiclepreparation upon lyophilization in the presence of a lyoprotecting agentand subsequent reconstitution in a pharmaceutically acceptable liquidcarrier.